telomerase peptide vaccination combined with temozolomide ... · lars julsrud2, and steinar...

Cancer Therapy: Clinical

Telomerase Peptide Vaccination Combined with Temozolomide:A Clinical Trial in Stage IV Melanoma Patients

Jon Amund Kyte1,3, Gustav Gaudernack3,4, Svein Dueland1, Sissel Trachsel3,Lars Julsrud2, and Steinar Aamdal1,4

AbstractPurpose: The study is a proof-of-principle trial evaluating toxicity, immune response, and clinical

response in melanoma patients after combined therapy with temozolomide and the telomerase peptide

vaccine GV1001. Our previous GV1001 trials showed immune responses in approximately 60% of lung or

pancreatic cancer patients.

Experimental Design: Twenty-five subjects with advanced stage IV melanoma (M1B or M1C) received

concomitant temozolomide and GV1001. Temozolomide was administered 200 mg/m2 orally for 5 days

every fourth week, and GV1001 as eight injections over 11 weeks. Immune response was evaluated by

delayed type hypersensitivity, T-cell proliferation, and cytokine assays. The immunologic responders

continued monthly vaccination.

Results: The treatment was well tolerated. A GV1001-specific immune response was shown in 18 of 23

evaluated subjects (78%). Patients developing long-term T-cell memory survived more than those rapidly

losing their responses. The immune response exhibited several characteristics of possible clinical sig-

nificance including high IFNg/IL-10 ratios, polyfunctional cytokine profiles, and recognition of naturally

processed antigens. Survival compared favorably with matched controls from a benchmark meta-analysis

(1 year: 44% vs. 24%, 2 years: 16% vs. 6.6%). The clinical responses developed gradually over years,

contrary to what is expected from chemotherapy. Five patients developed partial tumor regression and six

more recorded stable disease. One patient has no remaining disease on fluorodeoxyglucose positron

emission tomography scans after 5 years.

Conclusions: The immunologic response rate is considerable compared with previous GV1001 trials

without concomitant chemotherapy, although low toxicity is retained. The results warrant further studies

of GV1001/temozolomide treatment and support the general concept of combining cancer vaccination

with chemotherapy. Clin Cancer Res; 17(13); 4568–80. �2011 AACR.

Introduction

Cancer chemotherapy had been to a large extent ineffec-tive until the development of broad regimes combiningdifferent agents. The limited efficacy of monotherapyreflects tumor heterogeneity and genetic instability, favor-ing escape of resistant tumor subpopulations. Cancer vac-cination offers a different angle of attack, as compared with

cytostatics, and may therefore be particularly effective incombinatory regimes. However, most chemotherapeuticagents are immunosuppressive, and it was long assumedthat vaccines should therefore not be combined withchemotherapy. More recent knowledge has challenged thisconventional wisdom (1, 2). In particular, it is discussedwhether chemotherapy may be utilized to counter tumorprotection from regulatory T cells (Tregs), while prevailingthe immunocompetence necessary for vaccine response(3–5).

Here we report a proof-of-principle trial (phase I/II) ofcombined therapy with temozolomide and telomerasepeptide vaccination in 25 stage IV melanoma patients.The primary objectives were toxicity and immunologicresponse. Tumor response was a secondary study objective.Current prognosis for metastatic melanoma is dismal, withmedian survival of 4 to 9 months in patients with visceralinvolvement (6, 7). Dacarbazine has been consideredstandard treatment, but the tumor response rate is only6% to 15%, mostly partial responses, and there is nodocumented effect on survival (7). The oral drug temozo-lomide is metabolized to dacarbazine in vivo and carries apotential advantage in penetration of the blood–brain

Authors' Affiliations: 1Section for Clinical Cancer Research, Departmentof Oncology and 2Department of Radiology, Oslo University Hospital;3Department of Immunology, Cancer Research Institute, Oslo UniversityHospital and University of Oslo; and 4Faculty of Medicine, University ofOslo, Oslo, Norway

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

Note: The study was presented in part as abstract at ASCO 2006 (abstract8031).

Corresponding Author: Jon Amund Kyte, Department of Oncology, OsloUniversity Hospital, Radiumhospitalet, 0310 Oslo, Norway. Phone: 47-22-93-40-00; Fax: 47-22-93-58-08; E-mail: [email protected]

doi: 10.1158/1078-0432.CCR-11-0184

�2011 American Association for Cancer Research.

ClinicalCancer

Research

Clin Cancer Res; 17(13) July 1, 20114568

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barrier. In phase III trials, temozolomide and dacarbazinehave produced similar response rates (8, 9).Telomerase represents a possible target for universal

cancer vaccines (10, 11). The enzyme maintains telomerelength in dividing cells and is considered essential fortumor growth. Telomerase activity has been shown in allstudied cancer forms, including stem cell–like tumor cells(12–14). Interesting results have been reported fromphase I/II studies with 3 telomerase peptide vaccines(15–20). Peptide GV1001 represents a 16 amino acidsequence (EARPALLTSRLRFIPK) from the active site ofhuman telomerase reverse transcriptase (hTERT; refs. 21,22). The peptide was selected on the basis of computeralgorithms predicting strong human leukocyte antigen(HLA) class II binding properties and multiple nestedHLA class I binding motifs. The GV1001 vaccine may thusrecruit both CD4þ T helper cells and CD8þ cytotoxic Tcells. We have previously conducted 2 phase I/II trialswith GV1001, in pancreatic and lung cancer patients (19,20). Both studies were conducted without chemotherapy.The results indicated no serious adverse effects, a GV1001-specific immune response in approximately 60% ofpatients and an association between immune responseand survival. T-cell clone studies showed both T helperand cytotoxic responses (19, 20, 23). In the present trial,we hypothesized that temozolomide may enhance theimmune response by inducing antigen release or mod-ulating tumor tolerance. The study, to our knowledge, isthe first clinical trial evaluating combinatory therapy withtemozolomide and a cancer vaccine.

Patients and Methods

PatientsTwenty-five subjects with stage IV melanoma were

enrolled between January 2004 andNovember 2005. Inclu-sion criteria: histologically confirmed melanoma, nonre-sectable metastatic disease (AJCC stage IV), any HLAgenotype, measurable tumor, age between 18 and 75 years,Eastern Cooperative Oncology Group (ECOG) perfor-mance status 0–2, adequate hematologic, renal, and hepaticfunction. Exclusion criteria: previous chemotherapy, clin-ical signs of brain metastases, severe cardiac disease, severeactive infections, and need for immunosuppressivemedica-tion. Screening for brain metastases was not done.

The trial was approved by the Norwegian MedicinesAgency, the Regional Committee for Medical ResearchEthics, and the Hospital Review Board. It was carried outin compliance with theWorldMedical AssociationDeclara-tion of Helsinki. Written informed consent was obtainedfrom all patients.

TreatmentThe treatment schedule is shown in Appendix Figure A1

(see supplementary data online). Temozolomide wasadministered according to standard recommendationsfor malignant melanoma, 200 mg/m2 orally on 5 conse-cutive days every 28 days. GV1001 was given as 3 injectionsduring week 2 (Monday, Wednesday, Friday), 2 injectionsduring week 3 (Monday, Friday), and single injections atweeks 6, 7, and 11. Immune responders were given boostervaccines at 4-week intervals. GV1001 (300 nmol peptide in125 mL saline) and adjuvant granulocyte macrophage col-ony stimulating factor (GM-CSF; 75 mg) was administeredby intradermal injection in the right paraumbilical area.

The duration of treatment depended on the clinical andimmunologic response, according to the protocol: patientswith objective tumor response or stable disease (SD) atweek 12 and immunologic response received additionalcycles with temozolomide and GV1001, with responseevaluation at every third cycle. Patients with objectiveresponse/SDwithout immunologic response received addi-tional cycles with temozolomide only. Patients with pro-gressive disease (PD) and immunologic responsediscontinued temozolomide, but were offered boostervaccination with GV1001 every 4 weeks. All patients com-pleting the first 7-week sequence of vaccines (23 of 25patients) were considered evaluable for immunologicresponse.

Clinical evaluationTumor response was assessed by computed tomography

(CT) scans and clinical examination at 12-week intervals,and classified according to the Response Evaluation Cri-teria in Solid Tumors (RECIST; ref. 24). Tumor responseand survival was evaluated by intention-to-treat analysis.

Blood screening and assessment of adverse drug reac-tions were conducted at each visit. Adverse events weregraded according to the National Cancer Institute common

Translational Relevance

Cancer vaccination and chemotherapy attack thetumor from different angels and may synergize to over-come tumor escape. Chemotherapy may facilitateimmunization by provoking inflammation and elimi-nating regulatory T cells. The present trial evaluatedcombined therapy with temozolomide and the telomer-ase peptide vaccine GV1001 in advanced melanomapatients. The results showed that 18 of 23 subjectsdeveloped a specific immune response, without notabletoxicity. Patients developing long-term immunologicmemory survived more than those rapidly loosing theirresponses. Survival was extended compared withmatched controls from a benchmark meta-analysis (1year: 44% vs. 24%). Five patients developed partialtumor regression, including 1 survivor that is negativeon positron emission tomography–computed tomogra-phy scans after 5 years. The 78% immune response rateis considerable compared with most vaccine trials,including previous GV1001 studies without concomi-tant chemotherapy. The results warrant further studiesof GV1001/temozolomide treatment and supportthe concept of combining cancer vaccination withchemotherapy.

Telomerase Vaccine and Temozolomide in Stage IV Melanoma

www.aacrjournals.org Clin Cancer Res; 17(13) July 1, 2011 4569




toxicity criteria (NCI-CTC) version 3.0 and consideredrelated to treatment if the relationship was reported asprobable or suspected.

Immunologic evaluationPeripheral blood mononuclear cells (PBMC) were

obtained at weeks 0, 5, 9, and 12 and at every third boostervaccination. Pre- and postvaccination samples were ana-lyzed in parallel for proliferation response to GV1001peptide stimulation (3H-Thymidine assays). T-cellresponses were considered GV1001-specific when the sti-mulatory index (SI; response with antigen divided byresponse without antigen) was more than 2. Specificimmune responses were further characterized by cytokineassays and T-cell clone experiments (HLA restriction, spe-cificity for truncated peptides, response to recombinanthTERT protein). Materials and methods for immunologicassays, delayed type hypersensitivity (DTH) recording, flowcytometry, and production of peptides and recombinanthTERT protein are described in Appendix 1 (see supple-mentary data online).

StatisticsThe study reports overall survival (OS) and progression-

free survival (PFS), calculated from start of study treatment.OS for the intention-to-treat population, calculated by theKaplan–Meier method, was compared with predicted sur-vival based on a meta-analysis of clinical trials including2,100 patients (6). The meta-analysis was conducted byKorn and colleagues for providing survival benchmarks forsmall scale trials. We carried out the calculation as recom-mended (6). Briefly, prognostic factors (sex, ECOG status,metastatic category) from our patients were inserted into amathematical formula generating predicted survival. Thecalculation was based on trials where brainmetastases werenot excluded, because we did not conduct screening forbrain involvement (see Discussion). A 95% CI was gener-ated for survival of the study group (n ¼ 25), whereas thepredicted survival curve was regarded as a fixed estimate(Fig. 3). Survival after 1 year for the study group wascompared with predicted survival by use of Student’s t test.

Results

Patient characteristicsTwenty-five patients with advanced stage IV melanoma

were enrolled (Table 1). The majority (16 of 25) had M1Cdisease (visceral metastases or elevated lactate dehydrogen-ase); the remaining 9 patients hadM1B disease (pulmonarymetastases). Metastatic sites, age, sex, and treatment detailsare listed in Appendix Table A1 (see supplementary dataonline).

ToxicityThe combined therapy with temozolomide and

GV1001 was well tolerated. One patient developed tran-sient neutropenia (CTC grade IV) and thrombocytopenia(grade III), considered related to temozolomide. Other-

wise, no treatment-related grade III/IV toxicity wasobserved. Milder side effects were recorded in all patients,most commonly representing nausea, vomiting, fatigue,or local skin reactions at the injection site. Allergic reac-tions (grade II) were observed in 2 subjects. Finally, therewas no evidence of long-term toxicity in follow-up sam-ples from patients with extended survival, in spite ofdurable GV1001-specific responses and repeated boostervaccination (maximum observation 5 years, see the fol-lowing text).

Induction of immune responsesT-cell proliferation assays were carried out on pre- and

postvaccination PBMCs from 23 of 25 patients, that is, inall patients whose relevant PBMCs were obtained. AGV1001-specific T-cell response was shown in 18 of 23(78%) of patients after vaccination (Fig. 1A). Six subjectshad a GV1001-response prior to vaccination (Fig. 1A),suggesting spontaneous priming against naturallyexpressed GV1001 or cross-reacting epitopes. The SIincreased substantially after vaccination even in these 6patients (2.1!79; 3.2!35; 4.6!29; 7.5!24; 11!116;26!122). Among those developing de novo responses,40% developed a response by week 5 and 90% by week 9.

The DTH reactions were negative during standard studytreatment in all subjects whose recordings were obtained,including 11 immune responders in T-cell assays. Interest-ingly, 3 patients turned positive after omission of temozo-lomide and continuous booster vaccination. In ourprevious GV1001 trials without temozolomide (19, 20),the DTH reactions were positive in a substantial number ofpatients (26 of 62). These observations may point to amodulating effect of temozolomide on the GV1001response. DTH reactions have been associated with Th-1profiles, but cytokine analyses showed that the presentDTH negativity did not reflect low-level Th1 cytokines(see the following text).

Development of long-term T-cell memoryMost cancer vaccine trials have for practical reasons been

limited to short-term vaccination and immunomonitoring,whereas clinical efficacy probably requires long-term T-cellresponses (25, 26). Here, immune responders were offeredcontinued monthly vaccination. Long-term follow-uprevealed that 10 of 12 patients continuing vaccinationdeveloped durable GV1001-specific T-cell activity. Strik-ingly, all the 10 patients with durable T-cell responses atmonth 6 survived more than the 2 patients rapidly losingtheir responses (Table 1). Moreover, the survivors exhibitedretained responses in follow-up samples obtained at latertime points, ranging from week 36 to week 258 (Fig. 1B;Table 1; data not shown).

Tumor responseAll 25 patients enrolled had PD at study entry and

belonged to the most advanced categories within stageIV (64% M1C; 36% M1B; 0% M1A). Interestingly, 5 sub-jects developed partial tumor regression (partial response;

Kyte et al.

Clin Cancer Res; 17(13) July 1, 2011 Clinical Cancer Research4570




PR). Their tumor volumes decreased by 65% to 96%(Fig. 2A). An additional 6 patients experienced diseasestabilization, whereas 14 subjects had continued PD. All5 patients with PR developed GV1001-specific T-cellresponses, and the immune responses prevailed through-out their tumor regression periods (Fig. 1B). Interestingly,the clinical responses developed far more gradually thanexpected from chemotherapy, with only 1 reaching PR atweek 12, 3 more within week 36, and the fifth at week 48.Patient 19 (P19) had disseminated MIC melanoma at

study entry, including multiple visceral metastases. After

start of vaccination, no new lesions appeared. The totaltumor diameter started decreasing gradually, reached PR atweek 48, and has continued to regress over a periodexceeding 5 years (Fig. 2B). Remarkably, the patient hasno symptoms of tumor disease, and all residual CT-lesionsare negative on fluorodeoxyglucose positron emissiontomography (FDG-PET) scans. As melanomas are highlyPET-sensitive, the patient may have a complete response(CR). She currently receives booster vaccines at 3-monthintervals and retains a strong GV1001-specific immuneresponse (Fig. 1B).

Table 1. Disease stage, immune response, and clinical outcome

Patient AJCCstagea

T-cellresponseb

SI aftervaccc

Long-termT-cell memoryd

Tumorresponsee

PFS(days)f

Survival (days)g

P1 IV; M1C NE NE NA PD - 68P2 IV; M1C POS 14 POS (>6 mo) SD 168 344P3 IV; M1C POS 11 NA PD - 257P4 IV; M1C NE NE NA PD - 84P5 IV; M1C POS 122 NA PD - 583P6 IV; M1B POS 35 POS (>9 mo) SD 168 379P7 IV; M1B POS 26 POS (>9 mo) PD - 317P8 IV; M1C POS 3.7 NEG (<6 mo) SD 140 289P9 IV; M1C POS 39 NEG (<6 mo) PD - 283P10 IV; M1C POS 12 NA PD - 194P11 IV; M1B POS 11 POS (>25 mo) PR 585 1,134P12 IV; M1B POS 8.0 NA PD - 151P13 IV; M1B POS 79 POS (>14 mo) PR 502 563P14 IV; M1C NEG 0.8 NA PD - 99P15 IV; M1C NEG 1.3 NA PD - 250P16 IV; M1C POS 15 POS (>10 mo) PR 385 795P17 IV; M1B NEG 1.3 NA PD - 1,998þ (alive)h

P18 IV; M1C POS 17 NA PD - 239P19 IV; M1C POS 61 POS (>60 mo) PRi 1,905þ 1,905þ (alive)P20 IV; M1C POS 24 POS (>9 mo) SD 168 590P21 IV; M1B POS 29 POS (>13 mo) PD - 413P22 IV; M1C POS 122 POS (>14 mo) PR 248 594P23 IV; M1B NEG 1.1 NA SD 168 312P24 IV; M1C POS 116 NA PD - 418P25 IV; M1B NEG 1.1 NA SD 154 169

0 M1A 18/23 10/12 5 PR Mean: 4979 M1B 6 SD Median: 31716 M1C 14 PD

aDisease stage and metastasis category (within stage IV) at study entry.bPOS, positive, that is, T-cell proliferation specific for GV1001 (SI > 2); NEG, negative (no specific response); NE, not evaluable, that is,first round of vaccination (7 injections) not completed and postvaccination samples not available.cSI (response with GV1001 divided by response without GV1001) after vaccination (vacc).dLong-term T-cell response. POS, GV1001-specific response > 6 months (last available and positive sample is indicated in brackets);NEG, turning negative within 6 months; NA: not applicable (because no initial T-cell response or no available long-term samples).eTumor response (RECIST). Best response during vaccination (compared with baseline, evaluated at 12-week intervals).fPFS from start of study treatment. CT scan at study entry was used as reference.gSurvival from start of study treatment.hP17 was included in a different phase I study and developed clinical response.iP19 has some residual lesions on CT, but these lesions are negative on FDG-PET.






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Figure 1. GV1001-specific T-cell responses. PBMCs were obtained prior to start of therapy, at weeks 5, 9, and 12 and at every third booster vaccination. ThePBMCs were stimulated once in vitro and tested for proliferation against irradiated PBMCs with or without peptide GV1001. A, pre- and postvaccinationT-cell response from all evaluated patients. Columns represent mean SI, that is, response with antigen (GV1001) divided by response without antigen.Responses with SI > 2 were considered GV1001-specific. B, long-term T-cell memory in clinical responders. Columns represent mean cpm of triplicates.Tumor response periods are indicated with horizontal bars below charts. The assays show durable GV1001-specific T-cell responses in 5 patients withPR and 1 with SD.

Kyte et al.





Figure 2. Tumor response. A, PRin 5 patients. Top, columnsrepresent total volume of allmeasurable lesions at study entryand minimum volume aftervaccination. Bottom, response ofindividual tumor lesions for eachpatient. Location of lesion: LH,liver hilus; LN, lymph node; Ma,mammary glands; Med,mediastnum; Mu, muscle; P,pulmonal; SC, subcutaneous Vvisceral. B; tumor response inpatient 19. Top, total diameter ofall measurable lesions(corresponding to RECIST) at timepoints for CT evaluation. P19stopped temozolomide at month8, but still receive boostervaccines at 3-month intervals.Middle, response of individuallesions at different time points. Allresidual CT lesions are negativeon FDG-PET scans, suggestingthat no viable tumor tissue mayremain. PET scans have not beenrepeated after month 42. Bottom,abdominal CT scans at study entryand after 61 months, showingregression of 3 metastatic lesions(CR ¼ complete regression; PR ¼partial regression). The month 61scan was taken without contrastdue to allergy.

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Patient 11 (P11) had multiple metastases to lung andmammary glands at study entry. CT-evaluations showed a29% regression in total tumor diameter at week 12,improving to 74% at week 36. All 5 metastases regressedpartly or completely [Fig. 2A; Appendix Fig. A3 (see sup-plementary data online)]. We recorded PD at week 84 dueto a new subcutaneous lesion, but continued vaccinationand temozolomide. The patient stayed in good generalhealth for 3 years, before developing brain metastases.Subjects P13, P16, and P22 also responded with gradualtumor regression, accompanied by durable hTERT-specificT-cell responses (Figs. 1B and 2A). CT scans showed regres-sion of 6 of 8, 4 of 4, and 7 of 7 prestudy lesions,respectively [Fig. 2A; Appendix Figs. A2 and A3 (see sup-plementary data online)].

Progression-free and overall survivalTable 1 lists PFS and OS, calculated from start of study

treatment. By intention-to-treat analysis (n ¼ 25), medianOS was 10.4months (range, 2.2–66months) andmeanOS16.3 months (M1B 20 months; M1C 14months). Immuneresponders had increased median OS and PFS comparedwith nonresponders (median OS 396 days vs. 250 days;median PFS 112 days vs. 84 days). These differences did notreach statistical significance (log-rank test; P > 0.05), reflect-ing the low number of subjects. Advanced melanomapatients usually progress further within weeks or fewmonths (6, 8). It is therefore of interest that the 5 PRpatients exhibited extended PFS ranging from 8 to morethan 62 months, as well as OS ranging from 19 to morethan 62 months.

Trial subjectsmay differ fromother patients, for example,due to selection processes determining which individualsare referred to trial units. Korn and colleagues have con-ducted a meta-analysis of 2,100 trial patients and suggested

that survival in phase I/II melanoma studies is comparedwith their data set (6). They also outlined a method forcalculating predicted survival, correcting for prognosticfactors. Applying this method, we calculated a survivaladvantage for this study treatment, bordering95%statisticalsignificance (Fig. 3; intention-to-treat analysis). The appar-ent advantagewas relatively stable at different timepoints (1year: 44% vs. 24%; 2 years: 16% vs. 6.6%; 3 years: 12% vs.3.7%). Moreover, one may note that the "control group"was not based on untreated subjects, but on trial patientsreceiving potentially active therapy. Korn and colleaguessuggested declaring a treatment worthy of further study ifthe 1-year survival was better than predicted, with a P valueless than 0.10. This criterion was met both by intention-to-treat and per protocol analysis (P ¼ 0.059 and P ¼ 0.032,respectively). The interpretation of these findings is stillcomplex, as discussed in the following text.

T-cell clones from clinical respondersWe generated 8 GV1001-specific T-cell clones from 2

clinical responders (P11 and P22). The clones were HLAclass II DR restricted (Fig. 4A; data not shown). Takentogether with our data from other studies (refs. 19, 23;unpublished data), we find that GV1001 is recognized on aseries of DP, DR, and DQmolecules. The HLA promiscuityof GV1001 suggests that the vaccine is applicable to thegeneral patient population, without a need for HLA typing.

A diverse response in terms of HLA restriction and finespecificity may reduce the risk of tumor escape (25). Here,we determined the fine specificity of T-cell clones by experi-mentswith truncated peptides spanning theGV1001 aminoacid sequence. The 4 analyzed clones from subject P22recognized 3 different core motifs from the N-terminal partof GV1001 (Fig. 4B). One of themotifs was shared by a P11clone (Fig. 4D). In previous studies, we have observed thatmultiple motifs from the central and C-terminal parts ofpeptide GV1001 may also be recognized (21).

Vaccine-specific T-cell clones may be of sparse relevancein vivo if they fail to recognize naturally processed antigens.Here,weobserved recognitionofnaturally processedhTERTepitopes (Fig. 4C and D). Most clones responded morevigorously to hTERT protein than to pure GV1001 peptide(Fig. 4C; data not shown). We also noted strong responsesagainst low concentrations of truncated peptides (Fig. 4CandD). These observations suggest that the clonesmayhavehigher affinity for naturally processed epitopes than for thevaccine peptide. The data also point to a simplemechanismfor epitope spreading, as the cross-reacting natural epitopesmay stretch beyond the GV1001-sequence.

Cytokine profilesThe cytokine profiles for 14 of 18 immunologic respon-

ders were investigated in Bioplex assays by using panelsmeasuring up to 27 cytokines. The results showedGV1001-specific secretion of multiple cytokines in all14 patients [Fig. 5; Appendix Table A2 (see supplemen-tary data online)]. According to the Th1/Th2-paradigm, aTh1-like pattern is desirable for cancer eradication (27).

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Figure 3. Survival in study patients compared with benchmark meta-analysis. Red line, OS for the study patients, calculated from start ofvaccination. Dashed red lines, 95% CI. Blue line, predicted survival for ourpatients (fixed estimate), calculated from Korn's meta-analysis ofmelanoma trials were brain metastases were not excluded (6). Thecalculation corrects for prognostic factors (performance status, site ofmetastases, etc.) and was done as recommended by Korn and colleagues.The analysis suggests a difference in favor of this study treatment,reaching a borderline statistical significance.

Kyte et al.





Figure 4. GV1001-specific T-cellclones from clinical responders.T-cell clone (T) proliferation afterstimulation with irradiated EBV-transformed cells (EB) with orwithout peptide GV1001 or hTERTprotein. Columns represent meancpm of triplicate wells. A, patient22. HLA restriction wasdetermined by blockage withmonoclonal antibodies againstDP, DQ, or DP molecules. B,patient 22. Fine specificityanalysis by stimulation withtruncated peptides covering theGV1001 sequence (amino acidsequences given in right text box).The 4 P22 clones recognize 3different core sequences,annotated in text boxes below thechart. C, patient 22. Recognitionof naturally processed antigens.T-cell clones were stimulated withEBV-transformed cells incubatedwith recombinant hTERT protein.The assay indicates response tonaturally processed protein inclones 5, 10, 18 (not 45). D, patient19. Fine specificity andrecognition of naturally processedantigens. T-cell clones werestimulated with truncatedpeptides or EBV-transformed cellsincubated with recombinanthTERT protein. The coresequence recognized ishighlighted in red below the chart.

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Figure 5. Secretion of Th1/Th2 cytokines in postvaccination T-cell cultures. Cytokine assays were carried out in 14 of 18 immune responders, that is, allsubjects were sufficient samples were obtained. T cells were stimulated with irradiated PBMCs with or without peptide GV1001. Supernatants wereanalyzed in duplicates or triplicates by Bioplex cytokine assays. Columns represent mean concentration (pg/mL). Data for non-Th1/Th2 cytokines are shown inAppendix Table A2 (see supplementary data online).

Kyte et al.





However, we have previously observed that humanresponses frequently do not follow a Th1/Th2 delineation(23, 28, 29). The present responses comprised high levelsof key Th1-cytokines IFNg and TNFa, but also of hallmarkTh2 cytokines, interleukin 5 (IL-5) and IL-13. Thisapplied both to the T-cell bulk cultures (Fig. 5) and tothe T-cell clones (ref. 23; data not shown). Collectively,our present and previous results indicate that cytokineprofiling should not rely on a Th1/Th2 dichotomy. Ofnote, we detected only low levels of IL-4 and IL-10(Fig. 5). The responses would therefore easily be desig-nated "Th1" with commonly used panels measuring onlyIFNg , TNFa, IL-4, and/or IL-10. Interestingly, the highIFNg/IL-10 ratios may reflect a favorable balance betweenimmunity and tolerance, as IL-10 is considered to pro-mote regulatory T cells and "tolerogenic" dendritic cells.The analyses further showed GV1001-specific secretion

of a broad range of proinflammatory cytokines and che-mokines [Appendix Table A2 (see supplementary dataonline)], including IL-1b, IL-6, IL-8, GM-CSF, and macro-phage inflammatory protein-1b. This polyfunctional cyto-kine profile was observed in all evaluated patients, both inT-cell bulk cultures and T-cell clones (Appendix Table A2(see supplementary data online); ref. 23; data not shown).We also noted that the GV1001-specific responses includedIL-17. A subset of activated T-cell clonesmay thus belong tothe Th17 lineage (30, 31).Finally, we asked whether temozolomide had affected

the cytokine profile of the GV1001 response and comparedthe cytokine patterns to data from patients in our otherGV1001 trials (Appendix Fig. A4 (see supplementary dataonline); refs. 20, 23; data not shown). The results suggestedno substantial difference in the range of cytokines secretedor the ratio between key cytokines.

Discussion

The concept of combining cancer vaccination with che-motherapy is attractive, and also challenging. Because ofthe long-standing assumption that chemotherapy wouldpreclude immunization, there is sparse experience on howthese modalities interact. Here, we combined temozolo-mide with an hTERT peptide vaccine. The lack of increasedtoxicity is of particular interest, as temozolomide is bonemarrow toxic and telomerase is expressed by hematologicstem cells. There is also no evidence of long-term toxicity insubjects receiving up to 5 years booster vaccination, in spiteof durable immune responses. Interestingly, the immuno-logic response rate of 78% is higher than that in anyprevious GV1001 trial (19–22, 32). Follow-up studies of12 immune responders revealed that 10 patients withdurable GV1001 responses all survived more than the 2subjects rapidly losing their T-cell activity. The clinicalevaluation showed objective tumor responses in 5 subjects.OS was extended compared with matched controls from abenchmark meta-analysis.The immune response rate of 78% indicates that temo-

zolomide did at least not preclude immunization. Most

subjects had normal lymphocyte counts at time of vaccina-tion (data not shown). This may be related to the temo-zolomide-free intervals in the dosage regime formelanoma. Temozolomide is known to affect T-cell counts,but mostly if administered continuously (33–36). Theconsiderable immune response rate may, moreover, reflecta beneficial effect of temozolomide. Several studies in micehave reported enhanced immunization after combiningvaccination with temozolomide, or that temozolomidesuppresses Treg function (4, 36–40). Chemotherapy mayalso suppress myeloid-derived suppressor cells (5).

The immunologic evaluation per protocol did notinclude Treg analyses, but we made an attempt to investi-gate the influence of temozolomide on Treg counts. Flowcytometry analyses showed that CD4þCD25high cells with aTreg phenotype were present in peripheral blood aftertemozolomide treatment and after the subsequent vaccineinjections (data not shown). We did not obtain sufficientpretreatment samples to determine whether temozolomidehad still affected Treg frequency and can therefore notmakeany conclusions about the effect of temozolomide on T-cellpopulations. In our ongoing melanoma vaccine trial(NCT00961844), this issue is addressed. Interestingly, pre-liminary data suggest that temozolomide inducesdecreased Treg counts (unpublished data). The cytokinedata reported earlier in the text indicated that temozolo-mide had not substantially altered the cytokine profilescompared with previous GV1001 trials. Nevertheless,temozolomide is known to induce apoptosis of melanomacells and is likely to induce inflammation and cross-prim-ing of tumor-associated antigens. We hypothesize that thisprocess may synergize with CD4þ T helper cells recruitedthrough GV1001 vaccination. T helper cells may in parti-cular engage antigen presenting cells (APC) presentingantigens from apoptotic tumor and induce epitope spread-ing (27). In ongoing studies of long-term survivors afterGV1001 vaccination, we have detected responses againsttelomerase antigens not included in the vaccine (unpub-lished data).

Although most peptide vaccines have represented shortCTL epitopes, the use of longer peptides recruiting T helpercells may yield advantages in vivo (1, 25, 27). A T helperresponse may more effectively synergize with apoptosisand inflammation induced by radiation or chemotherapy,as depicted earlier in the text. The durable memoryresponses reported herein for GV1001 point to anotherpossible advantage; several studies have suggested that Thelper activity is necessary for the development of CTLmemory (41, 42). Long peptides may, moreover, be pro-cessed by endogenous APCs and recruit T-cell clones withdiverse specificities, as observed for GV1001. Several longpeptides, including GV1001, have furthermore been shownto elicit cytotoxic responses against nested short epitopes(1, 19, 43–45).

The present GV1001 responders developed polyfunc-tional cytokine profiles with mixed Th1/Th2 patterns anda high IFNg/IL-10 ratio [Fig. 5; Appendix Table A2 (seesupplementary data online)]. The broad and proinflamma-






tory cytokine profile may mobilize the adaptive and theinnate immune system. Interestingly, polyfunctional cyto-kine profiles have been associatedwith protective immunityin patients surviving HIV or Leishmania (46–48). Polyfunc-tional cytokine patterns may be particularly important in acancer vaccine setting, where there is a need to overcomeestablished tumor tolerance and transform the inflamma-tory milieu.

Six patients in the present trial harbored T-cell responsesagainst GV1001 prior to vaccination, and others havereported spontaneous GV1001-specific activity in subjectswith chronic lymphocytic leukemia (49). Furthermore, weshow that GV1001-specific T-cell clones recognize endo-genous APCs pulsed with hTERT protein. Both the pre-vaccination reactivity and the hTERT protein responsessuggest that GV1001-associated epitopes are naturally pro-cessed and immunogenic in cancer patients. This study didnot include screening of tumors for telomerase expressionand hence did not address whether quantitative differencesin expression level between patients may have influencedthe vaccine response.

We report objective tumor regression in 5 patients andtemporary disease stabilization in an additional 6 sub-jects. Several observations suggest that the regressions areunlikely to be caused only by temozolomide. First, allclinical responders belonged to the immunologicresponse group and developed long-term GV1001-speci-fic memory. Second, the response rate is relatively highcompared with the approximately 13% rate reported inphase III temozolomide trials (8, 50). Third, chemother-apy responses rarely occur in patients such as P19 withwidespread visceral metastases outside of lung. Fourth,P19 developed most of her tumor regression after omit-ting temozolomide at month 8 and continuing vaccina-tion (Fig. 2B). Fifth, responses to temozolomide arefrequently short-lived, whereas 4 of 5 responders hereexhibited PFS more than 1 year. Finally, responses tochemotherapy usually materialize shortly after start oftreatment. By contrast, 4 of 5 responses in the present trialfell short of PR criteria at month 3, and all responsesgradually improved over months or years. This pattern isin accordance with vaccine responses, depending onmobilizing the immune system and overcoming tumortolerance. Taken together, these observations suggest arole of GV1001 in the development of clinical responses.There is, however, a need to conduct a randomized studybefore making any conclusions on clinical efficacy. Theslow development of tumor responses shows that a con-ventional 3-month evaluation on the basis of RECISTmay be misleading in cancer vaccine trials and supportsthe concept of developing adjusted response criteria, assuggested by others (26).

The group of study patients survived more thanexpected, compared with predicted survival as calculatedfrom Korn’s meta-analysis. This finding should be inter-preted with utmost caution, but is in line with possibleclinical benefits suggested by the tumor regression andPFS data. Subjects with clinical signs of brain metastases

were excluded from our trial. On the basis of the highfrequency of asymptomatic brain involvement amongpatients with disseminated melanoma, it is still likely thata considerable fraction carried brain metastases. Becausescreening for cerebral involvement was not done, we calcu-lated predicted survival on the basis of Korn’s data fromstudies not excluding brain metastases. Of note, patientswith clinical signs of brain metastasis are rarely enrolled intrials, and the calculation takes into account the generallygoodperformance status in our patients. Survival in our trialwas calculated from the start of study treatment, whereasKorn’s meta-analysis gives survival from a possibly earlierpoint ("time of registration"). The latter difference suggeststhat our survival advantage may be underestimated. In all,these issues illustrate that any comparisonbetweendifferenttrials carries considerable uncertainty. The apparent survivalbenefit ofGV1001/temozolomide is interesting, but need tobe reproduced with a randomized control group.

We conclude that combined therapy with temozolomideand GV1001 is well tolerated in advanced melanomapatients, and that standard temozolomide dosage for mel-anoma does not preclude development of vaccineresponses. The immunologic responses exhibit several fea-tures of possible clinical significance, including durable T-cell memory, recognition of naturally processed antigens,high IFNg/IL-10 ratios andpolyfunctional cytokineprofiles.Collectively, present and previous trials show that peptideGV1001may recruit a diverse spectrumof T-cell clones withdifferent HLA restrictions and fine specificity. Regardingclinical outcome, we observed objective tumor responsesin 5 long-term immune responders. OS was extended com-pared with predicted survival from a meta-analysis. Therehave also been signs of a possible clinical activity in ourprevious GV1001 studies (19–21). However, both clinicaland immunologic response rates are higher in the presenttrial, whereas the low toxicity fromGV1001monotherapy isretained. In our opinion, the results warrant further studiesof combinatory therapy with GV1001 and temozolomide.Our findings also support the general concept of combiningcancer vaccination with chemotherapy.

Disclosure of Potential Conflicts of interest

G. Gaudernack is a member of the advisory board for Kael-Gemvax(patent holders for GV1001). The other authors declare no conflict ofinterest.

Acknowledgments

We thank M. Cvancarova for valuable help on statistical analysis, and J.A.Eriksen andM.Moeller at Gemvax.We also thank doctors and nurses at OsloUniversity Hospital for excellent clinical follow-up and patient care, andespecially K. Øwre, M. Nyakas, I. Sve, and L. Hegg. Gemvax AS, Norway,supplied GV1001 free of charge.

Grant Support

This work was supported by the Norwegian Cancer Society.

Received January 21, 2011; revised April 28, 2011; accepted May 8, 2011;published OnlineFirst May 17, 2011.

Kyte et al.





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2011;17:4568-4580. Published OnlineFirst May 17, 2011.Clin Cancer Res Jon Amund Kyte, Gustav Gaudernack, Svein Dueland, et al. PatientsTemozolomide: A Clinical Trial in Stage IV Melanoma Telomerase Peptide Vaccination Combined with

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