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Published OnlineFirst August 24, 2010; DOI: 10.1158/1078-0432.CCR-10-1757

Clinical
Canceresearch
cer Therapy: Clinical

dritic Cell Vaccination in Combination with Anti-CD25oclonal Antibody Treatment: A Phase I/II Study in

R

astatic Melanoma Patients

es F.M. Jacobs1,2,3, Cornelis J.A. Punt1, W. Joost Lesterhuis1,2, Roger P.M. Sutmuller5,
ry-lène H. Brouwer2, Nicole M. Scharenborg2, Ina S. Klasen4, Luuk B. Hilbrands3, . Figdor2, I. Jolanda M. de Vries1,2, and Gosse J. Adema2
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pose: The success of cancer immunotherapy depends on the balance between effector T cells andessive immune regulatory mechanisms within the tumor microenvironment. In this study we inves-whether transient monoclonal antibody–mediated depletion of CD25high regulatory T cells (Treg)

able of enhancing the immunostimulatory efficacy of dendritic cell vaccines.erimental Design: Thirty HLA-A2.1+ metastatic melanoma patients were vaccinated with matureitic cells pulsed with tumor peptide and keyhole limpet hemocyanin (KLH). Half of the patientsretreated with daclizumab, a humanized antibody against the interleukin-2 (IL-2) receptor α-chain5), either four or eight days before dendritic cell vaccinations. Clinical and immunologic parametersetermined.ults: Daclizumab efficiently depleted all CD25high immune cells, including CD4+FoxP3+CD25high

from the peripheral blood within four days of administration. Thirty days after administration,umab was cleared from the circulation and all CD25+ cells reappeared. The presence of daclizumabdendritic cell vaccinations prevented the induction of specific antibodies in vivo but not the pres-f antigen-specific T cells. Daclizumab, however, did prevent these CD25+ T cells from acquiringr functions. Consequently, significantly less patients pretreated with daclizumab developed func-, vaccine-specific effector T cells and antibodies compared with controls. Daclizumab pretreatmento significant effect on progression-free survival compared with the control group.clusions: Although daclizumab depleted the CD4+FoxP3+CD25high Tregs from the peripheral cir-n, it did not enhance the efficacy of the dendritic cell vaccine. Residual daclizumab functionallyessed de novo induced CD25+ effector cells during dendritic cell vaccinations. Our results indicate
suppr
that for immunotherapeutic benefit of transient Treg depletion, timing and dosing as well as Treg spec-ificity are extremely important. Clin Cancer Res; 16(20); 5067–78. ©2010 AACR.

diseasregresreportImm

anoma is considered one of the most immunogenicof cancers. This is based on the following arguments:veral melanoma-specific antigens have been identi-, 2); (b) functional lymphocytes specific for melano-

e increased in melanoma patients (3); (c)lating agents can have a positive effect on

pandivorabmecherantat thecellstumoimmuIn m

in theand intionamelanducesthat C

ons: Departments of 1Medical Oncology, 2Tumord 3Nephrology, and 4Laboratory of Medicalegen Centre for Molecular Life Sciences, Radboudn Medical Centre, Nijmegen, the Netherlands; andn de Wittelaan, Mechelen, Belgium

ry data for this article are available at Clinical Cancerttp://clincancerres.aacrjournals.org/).

thor: Gosse J. Adema, Department of Tumor Immu-entre for Molecular Life Sciences/278 TIL, Post Boxegen, The Netherlands. Phone: 31-24-3617600; Fax:mail: [email protected].

0432.CCR-10-1757

ssociation for Cancer Research.

ls.org

Research. on November 12,clincancerres.aacrjournals.org from

e outcome (4, 5); and (d) spontaneous melanomasions with simultaneous onset of vitiligo have beened (6).unotherapeutic clinical trials have succeeded in ex-

ng melanoma-specific effector T cells in vivo, but fa-le outcomes are still limited because tumor-inducedanisms of immune evasion may render the host tol-for melanoma antigens (7, 8). Immunosuppressiontumor microenvironment mediated by regulatory T(Treg) is one of the most critical mechanisms ofr-immune escape and a major hurdle for successfulnotherapy (9–11).elanoma patients, selective accumulation of Tregsprimary tumor, tumor-infiltrated lymph nodes,metastases has been observed (12–14). The func-

l importance of these Tregs is shown in mouseoma models in which transient Treg depletion in-
antitumor immunity (15–19). These studies showD25 depletion effectively eliminates the CD25high
5067

2020. © 2010 American Association for Cancer

http://clincancerres.aacrjournals.org/

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Translational Relevance

Immunity against malignant cells depends on thebalance between the activity of immune effector cellsand immune regulatory mechanisms. CD25+Foxp3+

regulatory T cells (Treg) prevent immune attack by di-rectly suppressing the activation of immune effectorcells. The detrimental effect of Tregs in cancer has beenshown in multiple murine models in which transientdepletion of Tregs enhances antitumor immunity.

In this study, we treated metastatic melanoma pa-tients with the anti-CD25 antibody daclizumab priorto dendritic cell–based vaccination. We investigatedwhether the anti-CD25 antibody transiently depletedCD4+FoxP3+CD25high Tregs and whether this en-hanced the immunostimulatory efficacy of dendriticcell vaccines. Our results show that despite transientTreg depletion using anti-CD25 in patients, a bluntedrather than enhanced T-cell response to the vaccinewas observed. The data imply that for immunothera-peutic benefit, timing and dosing and especially theTreg specificity of the depleting compound are ex-tremely important.

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although an effect on CD25+ effector lymphocyteslso been reported. Apparently, CD25 depletionrarily resets the T effector/Treg balance in favorT effector subset. In mice several successful meth-gies of Treg depletion have been described (20).ed to great interest in the depletion of Tregs asf a multifaceted immunotherapeutic treatment ofoma patients. However, the ability to effectivelyate human Tregs in vivo has been limited by thef an exclusive cell surface marker. Tregs constitu-express high levels of the interleukin (IL-2) recep--2R; ref. 21). IL-2 delivers signals that are essentiale thymic development, peripheral survival, andonal fitness of Tregs (22–25). These observationsraised the question of whether drugs that interfereL-2 signaling might decrease the number of func-Tregs.ileukin diftitox (Ontak) is a recombinant IL-2/diph-toxin conjugate that is designed to direct the cyto-action of diphtheria toxin to cells that express the(26). Thus far, clinical studies in which Ontak wasistered to melanoma patients showed that it may re-he number of circulating Tregs and cause regressionlanoma metastases (27–29).pared with Ontak, monoclonal antibodies (mAb)t the IL-2R α-chain (CD25) are more effective in re-g Tregs in melanoma-bearing mice (20). In addition,D25 antibodies had a synergistic effect when com-with dendritic cell–based immunotherapy in mice
Daclizumab (F. Hoffmann-La Roche,) is a human-ntibody directed against CD25 (30).
urineavaila

ancer Res; 16(20) October 15, 2010

Research. on November 12,clincancerres.aacrjournals.org wnloaded from

he present study we investigated whether treatmentsingle low dose of daclizumab resulted in transienttion of Tregs in human melanoma patients andcement of the immunostimulatory efficacy of sub-nt dendritic cell vaccinations. We therefore vacci-30 HLA-A2.1+ metastatic melanoma patients withitic cells pulsed with tumor peptide and keyhole

d with daclizumab.

rials and Methods

t inclusionusion criteria were metastatic melanoma with mea-le disease parameters, expression of gp100 and tyros-in at least one metastasis, HLA-A2.1 phenotype,performance status 0 or 1, no second malignancy,ious concomitant disease, no concomitant treatmentmmunosuppressive drugs, and normal hepatic andfunction. Patients with central nervous system metas-were excluded. Prior treatment related to metastatice was allowed, provided a treatment-free period of atour months was observed. Patients with distant non-al metastases were categorized according to thecan Joint Committee on Cancer (AJCC) as M1a; pa-with metastases to the lungs were categorized asnd patients with metastases to any other visceral siteh elevated serum lactate dehydrogenase were catego-as M1c (31). All patients gave written informed con-rior to inclusion in the study. Approval from theregulatory committee was obtained.

al protocol and immunization scheduleeligible patients, a leukapheresis was done fromdendritic cells were generated (32). Antigen-

d mature dendritic cells were administered i.v.d. in close proximity to the inguinal lymph nodestimes biweekly. Patients in the daclizumab grouppretreated with 0.5 mg/kg daclizumab i.v. eithers (first cohort of 7 patients) or 8 days (second co-f 8 patients) before the first dendritic cell vaccina-Patients were treated in a nonrandomized fashion.the control group was included and treated, thenaclizumab group.linical response was defined as stable disease for ≥4hs or any partial or complete response. Response wasred by Response Evaluation Criteria in Solid Tu-(33). Observation of progressive disease led to cessa-f vaccinations. Progression-free survival (PFS) wasated from the day of the first vaccination. Vaccine-ic immune response was the primary end point.al response was the secondary end point.

le IL-2Rα measurementuble IL-2Rα (sIL-2Rα) levels were determined in
samples (U/mmol creatinine) using a commerciallyble immunometric assay (Immulite).
Clinical Cancer Research



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odies and immunostainingcharacterize the phenotype of the ex vivo generateditic cells and the immune-cell subpopulations in the pe-al blood, flow cytometry was done using either FITC-,erythrin (PE)- or allophycocyanin-conjugated mAbs.llowing mAbs were used: anti-HLA class I (W6/32),LA DR/DP (Q5/13), anti-CD80 (Becton Dickinson,),D14, anti-CD83 (both Beckman Coulter), anti-anti-CD25 (clone M-A251, no interference with da-ab binding to CD25), anti-CD56, anti-CD86 (BDingen,), anti-Foxp3 (eBiosciences), anti-gp100), anti-tyrosinase (Novocastra), and anti-CX3CR1). Daclizumab antibody was covalently labeled to488 fluor using the Alexa-488 protein labeling kiting to manufacturer's procedure (Invitrogen).

ritic cells: preparation, characterization, andof administrationdritic cells were generated as described previouslyBriefly, peripheral blood mononuclear cells (PBMC)solated by density gradient centrifugation (Pharma-0 minutes, 4°C, 2,100 rpm) after leukapheresis.s were washed and monocytes were isolated by ad-ce to plastic. Monocytes were cultured at 15 × 106

-cm2 tissue culture flasks (Costar) in 20 mL of IL-4U/mL; Schering-Plough International), granulocytecyte-colony stimulating factor (800 U/mL; Schering-h International) and 2%human serum (HS; bloodbanknland) containing X-VIVO15medium (BioWhittaker).y 6 half of the medium was replaced by autologousphage-conditioned medium enriched with 10 μg/mLglandin E2 (Dr. G.R. Adolf, Bender & Co.) and 10 ng/mor necrosis factor-α (TNF-α; kindly provided byolf Bender, Vienna, Austria). Cells were harvested on. This procedure gave rise to mature dendritic cells asby high expression levels of MHC class I and II, CD80,

, and CD86, and the absence of CD14 (data not shown).

reservation of PBMCs and dendritic cellsdendritic cells were cultured directly after leukapher-d frozen as immature or mature dendritic cells forple vaccinations. Dendritic cells and PBMCs wereusing a cryo 1°C freezing container (Nalgene), ing medium consisting of 50% X-VIVO-15 (5% HS),S albumin, and 10% DMSO. Cells were thawed in awater bath, after which the cells were washed once inedium and once in medium of room temperatureuse.

de pulsingdritic cells were pulsed with wild-type peptides,154-167, gp100280-288, and tyrosinase369-376. Pulsingone directly after harvesting or after thawing. Onay of vaccination we added peptides (50 μg/mL)minutes and kept dendritic cells at 37°C/5%

Thereafter, fresh peptides (25 μg/mL) were added,
endritic cells were kept at room temperature for an-60 minutes. After peptide loading, dendritic cells
TetrRosali

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washed once in 0.9% sodium chloride and resus-d in 100 μL (5 × 106 dendritic cells) for i.d. injection200 μL (10 × 106 dendritic cells) for i.v. injection.

ed-type hypersensitivitye week after the last of three dendritic cell vaccina-a delayed-type hypersensitivity (DTH) skin testone as previously described (34). Briefly, unpulseditic cells, dendritic cells pulsed with gp100, dendriticulsed with KLH and gp100, dendritic cells pulsedyrosinase, and dendritic cells pulsed with tyrosinaseLH (10 × 105 dendritic cells each) were injected i.d.skin of the back of the patients at four differentThe diameter (in millimeters) of induration wasred by a dermatologist after 48 hours, and an indu-of >2 mm was considered positive. From DTH sites,biopsies (6 mm) were obtained under local anes-

. Biopsies were cut in half; one part was for immu-tochemistry, whereas the other part was cut in smalland cultured in RPMI/7% HS supplemented with100 units/mL). Every 7 days, half of the mediumplaced by fresh IL-2 containing RPMI/7% HS. Afterweeks of culturing, T cells were tested for tetramerg, antigen recognition, and cytokine secretion.

ral response to KLHore each vaccination, 80 mL of blood were collectedmunologic monitoring. Antibodies against KLHmeasured in the serum of vaccinated patients bye-linked immunosorbent assay as previously de-d (35). Briefly, 96-well plates were coated overnightwith the protein KLH (25 μg/mL) in PBS (0.1 mL/After washing the plates different concentrations oft serum were added for 1 hour at room tempera-fter extensive washing, specific antibodies (againstotal IgG, IgG1, IgG2, and IgG4) labeled with horse-peroxidase were allowed to bind for 1 hour at

temperature. Peroxidase activity was revealed using,5-tetramethyl-benzide as substrate and measured inrotiter plate reader at 450 mm.

erative response and cytokine productionHlular responses against KLH were measured in a pro-ion assay. Briefly, 1 × 105 PBMCs, isolated frombloodes taken before each vaccination, were plated per well6-well tissue culture microplate either in the presenceor without. After 16 hours of culture, supernatants

L) were taken, and the levels of IL-2, IL-4, IL-5, IL-10,, and IFN-γweremeasured by a cytometric bead arrayTh2 Cytokine CBA 1, BD PharMingen) according toanufacturer's instructions. After 4 days of culture,well of tritiated thymidine was added. Incorporationiated thymidine was measured in a β-counter.

tetramer staining
americ-MHC complexes were kindly provided by Dr.e Luiten and Dr. Hergen Spits from the Netherlands
Clin Cancer Res; 16(20) October 15, 2010 5069



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r Institute, Amsterdam, the Netherlands. Each tetra-as validated by staining against a CTL line specificLA-A2 in association with the peptide of interest.s (1 × 105 cells in 10 μL) were incubated withbeled tetrameric-MHC complexes for 1 hour attemperature. After washing, the samples were ana-by flow cytometry.

ine-secretion assayability of DTH–site infiltrating lymphocytes to rec-e vaccine-specific antigens and produce cytokineseasured in response to T2 cells pulsed with vaccine-ic peptides or the irrelevant HLA-A2.1 bindinge G250. To test for recognition of endogenouslysed antigens, DTH-infiltrating lymphocytes were in-ed with transfected HLA-A2.1-positive BLM targetexpressing either control antigen G250 or the anti-f study, gp100 or tyrosinase. Cytokine productioneasured in supernatants after 16 hours by cytometricarray as described above.

ntibody screeningwere screened for antinuclear antibodies by indirectnofluorescence on HEp-2000 cells in a serum dilu-f 1:40, according to the manufacturer's protocoledical Diagnostics). Sera were measured prior tof the vaccinations and after the third vaccination.

tical analysislan-Meier probability estimates of overall survivalalculated. The log rank test was used to determineical significance. Paired t-tests were done to test forent-related fluctuation of immune cells in the pe-al blood. Differences in KLH-specific proliferationLH-specific antibodies were analyzed by ANOVA.tistical tests were two-sided, and significance was de-ed as P < 0.05.

lts

t characteristicsrty HLA-A*0201-positive patients with progressivetatic melanoma received either daclizumab com-with dendritic cell vaccine therapy (daclizumab; n = 15) or dendritic cell vaccine therapy alone (con-n = 15). Patient characteristics are shown in Table 1.ts in the daclizumab group were on average 50.9old (range, 25-69), which did not significantly differhe control group (mean age, 51.7; range 22-73). Theoups were also not significantly different concerningto female ratio, AJCC stage of the melanoma, andtherapies.

ity and side effectssevere toxicity (common toxicity criteria grade 3-4)red. Vaccine-related side effects seen in patients in
roups were mild fatigue, anorexia, fever, and erythe-the intradermal injection site. None of the side
ally ivaccin



lasted >2 days after a vaccination. No daclizumab-d toxicity was observed. During follow-up, we didbserve any clinical signs of autoimmune disease. Be-daclizumab-induced transient Treg depletion mayse the risk of inducing autoreactive antibodies, weically tested for the presence of antinuclear antibo-efore and after vaccination. In both groups thereo significant increase of antinuclear antibodies ob-(Supplementary Table S1). During follow-up, onet in each group developed vitiligo.

ng of daclizumab to different blood subsetstest for the capacity of daclizumab to bind to differ-ood subsets, we incubated directly labeled daclizu-to erythrocyte-lysed whole blood cells of healthyls (n = 3). Supplementary Fig. S1A shows that dacli-b does not bind to granulocytes (gate 1) or mono-(gate 2) in the peripheral blood. Daclizumab bindsroximately 9% of the lymphocytes (gate 3). Daclizu-oes not bind to CD20+ B cells, but binds to a smalln of CD56+ natural killer (NK) cells, CD4+ Th cells,D8+ CTLs, and to a large fraction of CD4+FoxP3+

Incubation of lymphocytes with both daclizumab-88 and non–cross-reactive anti-CD25-PE showed on-ouble-positive and a double-negative population,shows that daclizumab specifically and exclusivelyto CD25+ cells (Supplementary Fig. S1B).

zumab pharmacokineticspharmacokinetics of daclizumab administered tots with metastatic melanoma is not known. Becauseclearance of sIL-2Rα bound to daclizumab ised, measurement of sIL-2Rα concentrations in theis an excellent predictor for daclizumab concentra-n the serum (36). Analysis of urine samples showedhe sIL-2Rα concentration decreased to undetectableimmediately after daclizumab administration inpatients (P < 0.001, paired t-test). Concentrations-2Rα were restored in approximately 4 weeksA). Daclizumab administration caused a completepid depletion of all CD25high cells from the periph-lood (P < 0.001, paired t-test). Repopulation ofhigh cells occurred approximately 4 weeks afterumab administration (Fig. 1B), as shown by usingross-reactive anti-CD25-PE.

tion of Tregs in vivoclizumab treatment transiently depleted CD4+

highFoxP3+ cells from the circulation (Fig. 1C and0.001, paired t-test). The percentage of FoxP3+ cellslready significantly decreased 4 days after daclizu-dministration (Fig. 1E; 44% reduction after 4 days,.027, paired t-test). The CD25highFoxP3+ Tregs wereally depleted. Daclizumab did not effectively depletexP3+ cells with low or intermediate CD25 expres-although we cannot exclude that they were function-
mpaired by bound daclizumab. Dendritic cellations without daclizumab had no effect on the



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Co-10 M n S 8Co-11 M S 9Co-12 M S 7 . 5Co-Co-Co-

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ntage of circulating CD4+FoxP3+CD25high Tregs1C-E). Daclizumab administration did not signifi-affect the percentage of peripheral blood CD4+

lls, CD8+ CTLs, CD56+ NK-cells, and CD20+ Bdata not shown). Daclizumab did, however, tran-increase the regulatory fraction of the NK cells

6brightCX3XR1low cells; Supplementary Fig. S2). Thissistent with previous reports showing daclizumab-

were dnation

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ed regulatory NK-cell expansion in patients sufferingautoimmune disease (37, 38).

ral responses against KLH in serummoral responses against KLH were detected in serumients not pretreated with daclizumab. Total IgG titers

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Lung cut M1b S, Tx 3 5 + + − − 1. 56/ Lung M1b S , I 3 2 + + − n.t. 1. 50/ Intest ne, s.c. M1c S , I 3 6 + + − n.t. 4. 66/ LN M1a 2 3 + + + +++ 13 35/ Kidne , LN, spleen M1c 9 1 + − − − 4. 54/ Intest ne, s.c. M1c S , I 3 9 + + − − 1. 22/ Sc, te ticle M1c 3 1 − + − n.t. 4 59/ LN, s . M1c S , I 5 1 − + − n.t. 2 33/ LN M1a 4 4 + + − +++ 114
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reviations: LDH, lactate dehydrogenase; CTx, chemotherapy; cut, cutaneous; I, IFN-α; LN, lymph node; RF, radiofrequencytion; RTx, radiotherapy; S, surgery; n.t., not tested.clizumab patients 1 to 7 received daclizumab 4 days before the first dendritic cell vaccination and patients 8 to 15 receivedlizumab 8 days before the first dendritic vaccination.H-specific cellular proliferation and antibodies (total IgG) after the first vaccination.sence of tetramer-positive T cells in DTH is marked as +, presence of functional tetramer-positive T cells in DTH is marked++.ramer-specific T cells in the blood were also detected before vaccination.

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ations, the IgG antibody titers further increased; ANOVA Pafter 2 vacc. = 0.011; Pafter 3 vacc. = 0.002).subtype analysis of IgG antibodies we observed onlynd IgG2, indicating the presence of IFN-γ and henceresponse (data not shown; ref. 39). In contrast, in se-

cells; daclizumab pretreatment (○; n = 6) depletes CD4 FoxP3 CD25both control patients and patients pretreated with daclizumab.

f patients pretreated with daclizumab, IgG antibodiese with KLH remained absent after the first dendritic

couldTh cel



ccination. After additional vaccinations, a nonsignif-ncrease in KLH-specific antibodies could be observedse patients (Fig. 2). The effect of the anti-CD25 anti-which is still present in the serum of patients duringitic cell vaccinations (Fig. 1), on antibody production

E, longitudinal measurements of the percentage of CD4 FoxP3

Daclizumab-mediated immune effects in vivo. A, soluble IL-2Rα levels in urine decreased to undetectable levels in all patients immediately aftermab administration. Restoration of soluble IL-2Rα levels started three weeks after daclizumab administration. B, kinetics of CD25high cells (% of totalcyte fraction) in the blood after daclizumab administration. C, representative dot plots on the expression of CD25 (Y-axis) and FoxP3 (X-axis) onated cells in PBMC during immunotherapeutic treatment. D, longitudinal measurement of the CD25high:CD25intermediate ratio of CD4+FoxP3+ cells.of daclizumab infusion;▴, dendritic cell vaccinations. Dendritic cell vaccination alone (•; n = 6) does not affect the percentage of CD4+FoxP3+

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eration of PBMCs upon KLH stimulationspective of daclizumab treatment, all patients devel-strong proliferative responses against KLH already af-single dendritic cell vaccination (Fig. 3A). Duringpecific proliferation of PBMCs high levels of TNF-αFN-γ, but not IL-4, could be measured, indicating1 nature of the immune response (Fig. 3B). Thesehow that daclizumab does not affect the generationH-reactive T cells in vivo and these T cells are func-upon stimulation in vitro. To study whether thepecific T-cell activation also occurs in vivo in thece of daclizumab, the KLH-specific proliferative ca-of day-33 PBMCs was tested in the presence of au-us serum taken at different time points after in vivoumab administration. Figure 3C shows that the pro-ive capacity of day-33 PBMCs was almost completelyated when serum was added with high concentra-
of daclizumab (i.e., a few days after daclizumabistration). Even after 5 weeks the daclizumab con-
vaccinspecif

P = 0.011;vacc. = 0.002).

acrjournals.org


tion in the serum of this patient was high enoughuce proliferation with 40% compared with serumut daclizumab (i.e., day −4). No inhibition ofpecific proliferation was observed with serum froml patients (data not shown). These data suggest thatients pretreated with daclizumab, vaccine-inducedpecific CD4+ Th cells are generated but are function-paired as long as daclizumab is present.

tion of tumor-specific T cells in vivoetermine the effect of daclizumab on the presence ofr-associated antigen-specific CD8+ T cells, tetramerngs were done after three vaccinations (Fig. 4A andementary Fig. S3A). In peripheral blood, tetramer-ve cells could be seen in only two patients (Dac-4o-4). In patient Dac-4 the tetramer-positive T cellsalso detectable in the blood prior to dendritic cell
ations. In both groups, significantly more antigen-ic CD8+ T cells were found in short-term T-cell
Antibodies against KLHA, representative total IgGies against KLH in theof a control patientic cell vaccination (vacc)a patient pretreated withmab at day −4, and apretreated with daclizumab−8. B, results of total IgGKLH of all patientsted with either dendriticone (black bars), dendriticth daclizumab pretreatment(white bars), and dendriticth daclizumab pretreatment(grey bars). A significante in total IgG titers wasd only in control patients

after 2 vacc.




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es (2-4 weeks) from biopsies of positive DTH reac-In 2 of 10 tested control patients antigen-specific Tere detected. The antigen-specific CD8+ T cells intwo patients were fully functional and producedevels of IL-2 and IFN-γ upon antigen-specific stim-n (Supplementary Fig. S3B). In patients pretreatedaclizumab, we detected antigen-specific T cells in3 evaluable patients. Strikingly, in 4 of 5 patientstetramer-positive CD8+ T cells were completely non-nal and did not produce any cytokines upon antigen-ic stimulation. The gp100154-tetramer-specific cellstient Dac-8 produced low amounts of IL-2, IL-5,FN-γ upon stimulation with the gp100154 peptideot upon stimulation with the endogenously ex-d gp100 protein (Fig. 4B and C). In conclusion,e-induced CD8+ T cells in the daclizumab grouppresent after the dendritic cell vaccinations. In con-o the CD4+ Th cells, however, these CD8+ T cellsfunctionally impaired in vitro even in the absencelizumab.

al outcomesignificant difference was seen in PFS between
ts that received daclizumab at day −4 or day −81). Analyzed as one group, patients that received
dermirespo



umab prior to dendritic cell vaccinations had thePFS compared with patients that received onlyitic cell vaccinations (Fig. 5 and Table 1). Both thenomonitoring data and the clinical follow-up indi-at daclizumab administration prior to dendritic cellation does not enhance the efficacy of the vaccine.al in both the daclizumab group and the controlwas comparable with what has been reported inerature for a population of patients with metastaticoma (40). Although the number of patients in thiswith specific T cells was limited and did not allowical analysis, the data are in accordance with our pre-work (34) showing that those patients with func-l antigen-specific T cells after vaccination have anged PFS (Table 1). Despite the presence of specificin five patients within the daclizumab group, their

ity to respond to tumor cells most likely explains thef impact on PFS in these patients.

ssion

une-suppressive CD4+FoxP3+CD25high Tregs accu-e in primary and metastatic melanomas, which un-

Figof PvacandPBprothaaftevacprorepdenC,PBvacpreobt(pa

nes spontaneous and/or vaccnses against melanomas (12–

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KLH-specific proliferationCs before and aftertion. A, both controls (•)clizumab patient-derived(○) show a strong

ative response against KLHuld already be detectedsingle dendritic celltion. B, cytokinetion by PBMCs ofntative patients after threeic cell (DC) vaccinations.-specific proliferation ofafter three dendritic celltions (day 33) in thece of autologous serum

ine-induced immune14). The aim of this

cal Cancer Research

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Tumor-specific T cells in vivo in patients pretreated with daclizumab. A, flow cytometric tetramer analysis of T cells derived after three dendriticcinations from biopsies of positive DTH reactions to dendritic cells pulsed with gp100 peptides. Percentage of tetramer-specific CD8 cells is
d in the dot plot. IFN-γ (black bars) and IL-2 (white bars) production of T cells derived from DTH reactions from patient Dac-8 (this is onentative patient out of four; B) and patient dac-9 (C) after stimulation with nonspecific and specific stimuli.
Clin Cancer Res; 16(20) October 15, 2010acrjournals.org 5075

Research. on November 12, 2020. © 2010 American Association for Cancerclincancerres.aacrjournals.org wnloaded from


studyclizumnomaon improof-melandendrdaclizdose dget effollowprovemunelympCD25signaleral sumousdepledies p19). Ter intdecreaWe

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was to investigate whether a single low dose of da-ab (0.5 mg/kg) transiently depletes Tregs in mela-patients and to assess possible effects of this drugmunity following dendritic cell vaccinations. In thisof-principle clinical trial we vaccinated 30 metastaticoma patients with peptide- and KLH-pulsed matureitic cells. Half of the patients were pretreated withumab, a humanized antibody against CD25. High-aclizumab (1 mg/kg biweekly) was designed to tar-fector lymphocytes to prevent acute graft rejectioning organ transplantation (41) and additionally

d effective for the treatment of patients with autoim-diseases or T-cell leukemia (42). Beside activatedhocytes, Tregs especially express high levels ofand are highly dependent on IL-2 (25). IL-2 deliverss that are essential for thymic development, periph-rvival, and functional fitness of Tregs (22–24). In

e melanoma models it has been shown that transienttion of Tregs using anti-CD25 monoclonal antibo-rovokes tumor-specific immune responses (15, 18,hese observations have raised the question of wheth-erference with IL-2 signaling via daclizumab mightse the number of functional Tregs (43).show that daclizumab efficiently depleted CD4++CD25high Tregs from the peripheral blood withins after administration. Approximately 30 days afterministration, daclizumab was cleared from the cir-n and CD4+FoxP3+CD25high Tregs reappeared. Im-response analysis revealed that significantly lessts pretreated with daclizumab developed functionale-specific effector T cells and antibodies comparedontrols. No antibodies against the “de novo antigen”ere present in the serum of daclizumab-treated pa-Subsequent in vitro analysis showed that in patients

tment with daclizumab (○). No significant difference between bothwas detected (P = 0.24, log rank test).

ated with daclizumab, vaccine-induced KLH-specificTh cells are generated and functional but only in the

whichTreg-d



ce of daclizumab. Vaccine-related tumor-associatedn-specific CD8+ T cells were detected in DTH biop-rom daclizumab-treated patients. However, theseailed to acquire effector function upon target cell rec-on even in the absence of daclizumab in vitro. Thesenomonitoring data suggest that in daclizumab-d patients the lack of proper Th-cell activation mayprevented effective generation of KLH reactive anti-s and vaccine-induced CD8+ T-cell responses, bothich depend on effective T-cell help. The direct impactlizumab on CD25+ B cells remains unclear.ltiple explanations for why daclizumab did notce or might even inhibit the immunostimulatory ef-of subsequent dendritic cell vaccines can be envis-Firstly, daclizumab does not deplete all FoxP3+

The residual CD4+FoxP3+CD25intermediate cells likelyent a functional (precursor) pool of Tregs. In addi-we cannot exclude that daclizumab-mediated CD4++CD25high-Treg depletion was less effective at thef the tumor and tumor draining lymph nodes. Sec-, daclizumab recognizes CD25 that is not exclusivelysed on Tregs (44). CD25 is known to be expressedtivated CD4 and CD8 T cells, but melanoma cellselves can also express low levels of CD25 (45, 46).ver, a single dose of daclizumab administered tooma patients transiently increases the number oftory NK cells that may have further compromisedficacy of the subsequent dendritic cell vaccinations.n vitro data show that daclizumab in the serum ofts disarms effector T cells. Aside from inhibitingnovo induction of effector T cells, daclizumab mayffect existing antitumor immune responses alreadyt in the patients. Previously, it had been reportedhe number of antigen-experienced CD25+ lympho-is greatly elevated in tumors and their drainingnodes (13). Because we show that daclizumab de-all CD25high lymphocytes in vivo, daclizumab mayneutralize this pre-existing antitumor immune re-e. A similar hypothesis was raised by Curtin et al.lain why anti-CD25 mAb in a mouse glioblastomal can eliminate newly induced tumors but cannot in-umor progression in established tumors (47). Third-all patients that received a single dose of 0.5 mg/kgumab, the drug was still present during subsequentitic cell vaccinations. Residual daclizumab titers mayrevented the induction of functional CD25+ effectory antigen-loaded dendritic cells. This may also ex-why depletion of Tregs by Ontak can enhance T-cellses upon vaccination in certain cancer patients (48).alf-life of this compound is very short, therefore thes no longer present at the time that vaccine-inducedr T-cell expansion occurs. In this context, it might besting to explore the original mouse mAb anti-Tac onhumanized daclizumab is based (49), as it will haveh shorter half-life than daclizumab in humans.r results indicate that immunotherapeutic trials in

rogression-free survival. Kaplan-Meier plot comparing the PFS oftic melanoma patients that were vaccinated with dendritic cells) and those that received dendritic cell vaccinations and

antibodies are used to target CD25, or relatedepleting strategies such as CD25-directed toxins




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9, 50), must carefully monitor the effects on all+, not Tregs only, and adjust timing and dosing sche-accordingly. A Treg-specific cell surface marker thatbe targeted with antibodies to specifically deplete/vate Tregs would overcome the daclizumab-relatedms encountered in this clinical trial.conclude that, although daclizumab depleted theFoxP3+CD25high Tregs from the peripheral circula-it did not enhance the efficacy of the dendritic celle. We show that residual daclizumab titers sup-d de novo induction of CD25+ effector cells duringitic cell vaccinations. Our results indicate that for im-therapeutic benefit of the transient Treg depletion,timing and dosing of daclizumab administrationtremely important. Alternatively, to overcome thenoregulatory influence of Tregs, novel approaches
cifically target and comprehensively eliminate or Rece
h) regulatory T cells are overrepresented in human metastaticlanoma lymph nodes and inhibit the function of infiltrating T cells.munol 2004;173:1444–53.

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osure of Potential Conflicts of Interest

otential conflicts of interest were disclosed.

owledgments

hank Dr. Strijk for doing the ultrasound guided, intranodal dendriticcinations and Dr. Gerritsen for the biopsies of the DTH site.

Support

Dutch Cancer Society (grant# KUN2003-2893), the Netherlandszation for Scientific Research (NWO-grant# 917-76363), the EU-Cancerimmunotherapy (LSHC-CT-2006-518234), and the EU-DC-THERA (LSHB-CT-2004-512074).costs of publication of this article were defrayed in part by thet of page charges. This article must therefore be hereby markedsement in accordance with 18 U.S.C. Section 1734 solely tothis fact.

ived 07/08/2010; accepted 08/09/2010; published OnlineFirst
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2010;16:5067-5078. Published OnlineFirst August 24, 2010.Clin Cancer Res Joannes F.M. Jacobs, Cornelis J.A. Punt, W. Joost Lesterhuis, et al. Metastatic Melanoma PatientsMonoclonal Antibody Treatment: A Phase I/II Study in Dendritic Cell Vaccination in Combination with Anti-CD25

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