Intralesional Treatment of Stage III Metastatic Melanoma ... · mulation of the payload at the site...

Research Article

Intralesional Treatment of Stage III Metastatic MelanomaPatients with L19–IL2 Results in Sustained Clinical andSystemic Immunologic Responses

Benjamin Weide1, Thomas K. Eigentler1, Annette Pflugfelder1, Henning Zelba2, Alexander Martens2,Graham Pawelec2, Leonardo Giovannoni4, Pier Adelchi Ruffini4, Giuliano Elia5, Dario Neri6, Ralf Gutzmer3,J€urgen C. Becker7, and Claus Garbe1

AbstractL19–IL2 is a recombinant protein comprising the cytokine IL2 fused to the single-chain monoclonal antibody

L19. In previous studies, intralesional injection with IL2 has shown efficacy for the locoregional treatment ofcutaneous/subcutaneous metastases in patients with advanced melanoma. The objectives of this study were toinvestigate whether (i) intralesional delivery of a targeted formof IL2would yield similar results, with reduction ofinjection frequency and treatment duration; and (ii) systemic immune responses were induced by the localtreatment. Patients with stage IIIB/IIIC melanoma and cutaneous/subcutaneous injectable metastases receivedweekly intratumoral injections of L19–IL2 at a maximum dose of 10 MIU/week for 4 consecutive weeks. Tumorresponse was evaluated 12 weeks after the first treatment. Twenty-four of 25 patients were evaluable for therapy-induced responses. A complete response (CR) by modified immune-related response criteria (irRC) of all treatedmetastases was achieved in 6 patients (25%), with long-lasting responses in most cases (5 patients for �24months). Objective responses were documented in 53.9% of all index lesions [44.4% CR and 9.5% partial responses(by irRC)], and 36.5% of these remained stable, while 9.5% progressed. Toxicity was comparable with that of freeIL2, and no serious adverse events were recorded. A significant temporary increase of peripheral regulatory T cellsand natural killer cells, sustained increase of absolute CD4þ lymphocytes, and decrease of myeloid-derivedsuppressor cells were observed upon treatment. Finally, we recorded encouraging data about the progressiontime to distant metastases and overall survival. Cancer Immunol Res; 2(7); 668–78. �2014 AACR.

IntroductionAdvanced cutaneousmelanoma has a poor prognosis, with a

median survival time of approximately 8 months (1) and about46,000 deaths per year worldwide (2). Stage III of the disease ischaracterized by the presence of metastases, limited to theanatomic region between primary melanoma and the nextproximal lymph node (LN) basin. It includes patients with LNand skin or subcutaneous metastasis (satellite and in-transitmetastases). Patients with locoregional satellite or in-transit

metastases are classified as N2c or N3 depending on theabsence or presence of concurrent or prior LN metastasis.According to the current American Joint Committee on Cancer(AJCC) classification, N2c patients are aligned to the clinicalstage IIIB in case of a nonulcerated primary melanoma, whileall other patients are classified as stage IIIC (3). Patients withdistant soft-tissue metastasis are classified as stage IV M1a ifserum lactate dehydrogenase (LDH) is within the normal rangeand other visceral metastases are absent.

Surgery is the first therapeutic option in these patients and isperformed with a curative intention in most cases. In contrast,options are few and of limited efficacy if surgery is not feasibledue to unresectable disease or continuous recurrences despiterepeated surgery. Until 2011, in addition to radiotherapy (4) andisolated limb perfusion (ILP; ref. 5), most of these patientsreceived systemic therapy with dacarbazine with palliativeintention. All of these treatments are characterized by lowefficacy. High-dose treatment with IL2 was approved by theU.S. Food andDrug Administration (FDA) in 1998 but, due to itsinherent toxicity, it found application only in a limited numberof young patients with excellent performance status (6). Thesituation improved after FDA approval of ipilimumab andvemurafenib, but the treatment-related survival benefit isrestricted to a minority of patients and long-term survival isstill rare.

Authors' Affiliations: Departments of 1Dermatology, Center of Dermato-Oncology and 2Internal Medicine II, Section for Transplantation Immunol-ogy and Immunohematology, University Medical Center, T€ubingen;3Department of Dermatology and Allergy, Hannover Medical School, SkinCancer Center, Hannover, Germany; 4Philogen S.p.A., La Lizza, Siena,Italy; 5Philochem AG, Otelfingen; 6Department of Chemistry and AppliedBiosciences, Institute of Pharmaceutical Sciences, ETH Zurich, Zurich,Switzerland; and 7Department of Dermatology and Venereology, Univer-sit€atsklinik Graz, Graz, Austria

Note: Supplementary data for this article are available at Cancer Immu-nology Research Online (http://cancerimmunolres.aacrjournals.org/).

Corresponding Author: Benjamin Weide, Department of Dermatology,Center of Dermato-Oncology, University Medical Center, Liebermeisterstr.25, D-72076 T€ubingen, Germany. Phone: 49-70712985748; Fax: 49-7071295265; E-mail: [email protected]

doi: 10.1158/2326-6066.CIR-13-0206

�2014 American Association for Cancer Research.

CancerImmunology

Research

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Intralesional therapy for satellite/in-transit melanomametastases has been proposed as an attractive therapeuticavenue with distinctive advantages over other approaches (7).Concentrations of the administered drug that can be reachedwithin the tumor are higher than those for systemic admin-istration, resulting in a beneficial therapeutic effect. Converse-ly, low systemic concentrations of the drug lead to reducedtoxicity. In contrast to surgery, diffuse metastatic spreaddistributed over large anatomic regions can be treated andpotential indirect beneficial systemic effects may result. Sev-eral cytokines (IFNa, IFNb, GM-CSF) have been used forintralesional treatment of metastatic melanoma with variableresults (8–12).Different groups, including ours, have reported the prom-

ising response of melanoma metastases to intratumoralinjection of IL2 (13–17). To improve this approach, we nowconsidered the use of antibody-based pharmacodelivery strat-egies. Cytokines of interest are expressed as fusion proteinswith antibody fragments, which allow the preferential accu-mulation of the payload at the site of disease while sparingnormal organs (18). We reasoned that a targeted form of IL2might reside in the metastatic lesion longer than the untar-geted form, thereby allowing a reduction in administrationfrequency and treatment duration.L19–IL2 is an immunocytokine, i.e., a recombinant fusion

protein of the monoclonal single-chain variable fragment(scFv) of antibody L19 and cytokine IL2. L19 recognizes thealternatively spliced extra-domain B (EDB) of fibronectin (FN),a marker of angiogenesis (19). EDB-containing FN is present inthe neovasculature of most solid tumors and hematologicmalignancies (19) but absent from almost all healthy adulttissues (except tissues of the female reproductive cycle).Therefore, L19–IL2 represents a targeted form of IL2, whichis able to accumulate at sites where angiogenesis occurs andthe EDB-FN antigen is expressed.L19–IL2 has already been studied in many preclinical mod-

els, and in patients, in phase Imonotherapy studies of renal cellcarcinoma (20) or in combination with dacarbazine in meta-static melanoma (21), with encouraging results.Here, we describe the results of a phase II clinical trial based

on the intralesional administration of L19–IL2 in patients withstage IIIB and IIIC metastatic melanoma.

Materials and MethodsPatientsThis multicenter study (ClinicalTrials.gov identifier:

NCT01253096) included patients from three clinical centers[University Medical Center (T€ubingen, Germany), HannoverMedical School (Hannover, Germany), and Universit€atsklinikGraz (Graz, Austria)] with approvals from the local ethiccommittees andnational authorities. The studywas conductedin accordance with the Declaration of Helsinki; all patientswere included after written informed consent. Patient inclu-sion criteria comprised histopathologically proven malignantmelanoma, presence of measurable and injectable soft-tissuemetastases in clinical stage IIIB or IIIC, male or female gender,with age �18 years, either without or after one line of prior

systemic treatment of metastatic disease, Eastern CooperativeOncology Group (ECOG) performance status <2, LDH < 2� theupper limit of normal, and a life expectancy of �12 weeks.Patients with evidence of visceral or brain metastases atscreening or severe cardiac disease (New York Heart Associ-ation grade > 2) or having undergone antitumor therapywithin4weeks of the administration of study treatment (exceptminorsurgery), and pregnant or lactating women were excluded.Concurrent treatment of metastatic melanoma was notallowed.

Study design and treatmentThe trial was an open-label, nonrandomized prospective

phase II study. Ten million international units of IL2 equiva-lents (Mio IU) of L19–IL2 (Darleukin; Philogen S.p.A.) wasdissolved in 6 mL glucose 5% supplemented with 0.2% humanserum albumin. Four milligrams of L19–IL2 corresponds to 24Mio IU of IL2 equivalents.

The treatment schedule was once weekly on an outpatientbasis. L19–IL2 was injected intratumorally using 30-gaugeneedles for superficial injections and 27-gauge needles fordeep injections, and the total daily dose was divided betweenall injectable lesions. All lesions present at screening wereinjected; a detailed list of lesions with their size and location isprovided in Supplementary Table S1. Sonography was used toguide injections of deep soft-tissue metastases. Patientsreceived analgesic treatment with acetaminophen or metami-zole 1 hour before treatment with L19–IL2 and received 0.5 to1.0 g of acetaminophen 5 or 10 hours after injection, as needed.

Tumor assessment was performed 2 weeks before the startof treatment (baseline) and at week 12. An additional tumorassessment was performed at week 6 after the first injection tocheck that no visceral metastases had developed under treat-ment. End-of-study (EoS) assessment was performed at week16 and follow-up for recurrence and survival every 6 weeksthereafter. Adverse events were graded according to the Com-mon Toxicity Criteria (version 3).

Response evaluationTheprimary endpointwas the rate of patientswith complete

responses (CR) at day 85, according to both immune-relatedresponse criteria (irRC; ref. 22) and Response EvaluationCriteria In Solid Tumors (RECIST) version 1.1 (23) modifiedas described below.

All lesions present at baseline were classified as index/nonindex lesions (irRC) or as target/nontarget lesions(RECIST). A lesion qualified as an index/target lesion by beingmeasurable as judged by the treating investigator. Lesiondiameters of �5 mm � 5 mm (irRC) or �5 mm (RECIST)were defined as cutoff points for measurability. Besides thelower cutoff size for RECIST (with respect to the cutoff size of�1.0 cm recommended in the original guideline; ref. 23), theassessment was not limited to five skin metastases, but includ-ed all the measurable injected lesions.

At later tumor assessments, the change in tumor burdenincluded the measurement of index and new measurablelesions, but all lesions (index, nonindex, new unmeasurable,and measurable lesions) were included in the overall modified

Intratumoral Treatment of Melanoma with L19–IL2, a Targeted Form of IL2

www.aacrjournals.org Cancer Immunol Res; 2(7) July 2014 669




irRC response assessment (22). For modified RECIST, thechange in tumor burden was based on the target lesions, butnew lesions and nontarget lesions were likewise considered fordefining the overall response (23).

In addition, all treated metastases (index and nonindexlesions) were evaluated individually for response. Index lesionswere measured at day 85 and EoS and classified as CR, partialresponse (PR), stable disease (SD), or progressive disease (PD)with respect to baseline. Response of injected nonindex lesions,which were almost exclusively <5 mm in size at baseline, wasclassified only as either CR (complete disappearance) or non-CR at day 85 and EoS.

Subcutaneous metastases were evaluated by sonography orCT scan.Whenever possible, in doubtful cases (e.g., if there wasresidual pigmentation), biopsies were taken for histopatho-logic confirmation of response.

Analysis of immune cell subsetsFor translational side studies, bloodwas drawn at baseline, 1

week after the last application of L19–IL2 (day 29), and attumor assessment (day 85). Peripheral blood mononuclearcells (PBMC) were immediately isolated from fresh blood byFicoll/hypaque density gradient centrifugation and cryopre-served until use.

After thawing, different populations of immune cells wereevaluated using multicolor flow cytometry. For the analysis ofCD4þ and CD8þ T cells, myeloid-derived suppressor cells(MDSC), regulatory T cells (Treg), and natural killer (NK) cells,Fc receptors were initially blocked with Gamunex (humanimmunoglobulin; Bayer), and dead cells were labeled forexclusion with ethidium monoazide (EMA; Invitrogen).

MDSCs were characterized by the CD14þCD11bþHLA-DR�/low phenotype (24–28) and NK cells as CD16þCD56low

(29). PBMCs were stained with CD3/PerCP, CD4/PerCP,CD8/PerCP, HLA-DR/PerCP-Cy5.5, CD11b/APC-Cy7 (BDBiosciences), CD56/Alexa Fluor 700 (BD Pharmingen),CD14/Pe-Cy7, and CD16/Pacific Blue (BioLegend). The per-centage of MDSCs was calculated as the proportion ofCD14þCD11bþHLA-DR�/low cells within the total popula-tion of viable PBMCs, while the percentage of NK cells wascalculated as the proportion of CD16þCD56low cells withinthe total population of lymphocytes.

For Tregs, which were characterized by the CD25þFoxP3þ

phenotype (30), cells were stained with CD25/PE, CD4/PerCP,and CD8/APC-H7 (BDBiosciences). After fixation and permea-bilization with Human FoxP3 buffer (BD Pharmingen), cellswere intracellularly stained with FoxP3/Alexa Fluor 647 (BDBiosciences). Frequencies of CD4þ and CD8þ T cells weredetermined within the total number of lymphocytes. Thepercentage of Tregs was calculated as the proportion ofCD25þFoxP3þ cells within all CD4þ cells.

NY-ESO-1– and Melan-A–specific T-cell responses weredetected as described previously (31). Influenza-specific T-cellresponses were also analyzed as a control. Briefly, cellswere stimulated with protein-spanning overlapping peptides(1 mg/mL; PepMix; JPT Peptide Technologies). After culturingfor 12 days, T cells were restimulated at a ratio of 1:2 withautologous, fluorescent-labeled PBMCs (10 mmol/L Cell Pro-

liferation Dye eFluor 450; eBioscience) either unpulsed (neg-ative control) or presenting one of the antigens in the presenceof Golgi-Plug (1 mL/mL; BD Biosciences) for 12 hours. AfterblockingwithGamunex and labelingwith EMA, cells werefixedand permeabilized with CytoFix/CytoPerm (BD Biosciences)and stained with CD3/eFluor605, IL10/PE, IL17/PerCP-Cy5.5(eBioscience), CD4/PerCP, IFN-g/PE-Cy7, CD8/APC-H7 (BDBiosciences), IL4/APC (BD Pharmingen), TNF/FITC, and IL2/Alexa Fluor 700 (BioLegend).

Samples were measured immediately using an LSR II andFACSDiva software (BD Biosciences). Antigen-specific Tcells were defined as being present if the following criteriawere met for at least one of the six analyzed cytokines: Thecytokine-producing cell population was clearly distinguish-able from nonproducing cells and the ratio between thepeptide-pulsed and unpulsed samples was greater than two.Only patients with detectable influenza-specific T cells wereconsidered to be evaluable for Melan-A– and NY-ESO-1–specific T cells. Data were analyzed using FlowJo software(TreeStar Inc.). A detailed illustration of the gating strategyfor the investigated immune populations is presented inSupplementary Fig. S2. Data interpretation for the analysisof antigen-specific T cells was performed as described indetail elsewhere (31).

Statistical analysisAll statistical analyses were conducted using the SPSS

software package Version 21 (IBM Inc.). Differences in meancell frequencies between different time points were calculatedwith the two-tailed paired t test. Differences in the proportionof patients with antigen-specific T cells and in response rateswere calculated using a two-tailed Fisher exact test. ForKaplan–Meier analyses, follow-up time for the calculation ofsurvival was defined from the date of first treatment either tothe date of the last follow-up or to the date of recurrence[recurrence-free survival (RFS)], diagnosis of the first distantmetastasis [distant metastasis-free survival (DMFS)] or death[overall survival (OS)], respectively. Disease-specific OS prob-abilities have been calculated, and only deaths due to mela-noma have been considered, whereas deaths due to othercauses were regarded as censored events. Estimates of cumu-lative survival probabilities according to Kaplan–Meier weredescribed together with 95% confidence intervals and com-pared using log-rank tests. Throughout the analysis, P values ofless than 0.05 were regarded as statistically significant.

ResultsPatients and treatments

Enrolment started on June 30, 2010, and was completed byAugust 23, 2012. The baseline data from the 25 patients aresummarized in Table 1. The median patient age was 68.4 years(range, 36.5–90.5 years). Most patients were treated in stageIIIC (21), and 4 patients were treated in stage IIIB. All metas-tases were accessible and injected in all patients.

Apart from surgical resection of the primary tumor, 17patients (68%) had undergone surgery for locoregional recur-rences. Thirteen of 17 patients had undergone two or more

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Cancer Immunol Res; 2(7) July 2014 Cancer Immunology Research670




previous surgeries, with the last surgery having been carriedout �8 weeks before the start of L19–IL2 therapy in 5 of thepatients. In 4 patients, limb perfusion or radiotherapy had alsobeen performed.Previous systemic treatments with IFNa (12 patients), che-

motherapy (3 patients), or both modalities subsequently (1patient) had been applied before L19–IL2 treatment wasinitiated. The median number of metastases treated perpatient was 5.5 (range, 1–118).

ToxicityAll 25 patients were included in the analysis of toxicity. The

treatmentwas generallywell tolerated, withmostly grade 1 and2 toxicities recorded. In 76% of patients, intratumoral L19–IL2therapy caused an inflammatory injection site reaction (localswelling and erythema) limited to the tumor tissue, reaching ina few cases to grade 3, followed by a selective tumor necrosisthat generally did not affect the surrounding normal tissue.Injection pain was present (only one grade 3 case), manageableby the application of a local anesthetic cream and oral meta-mizole. Twenty percent of patients experienced low-gradefever, easily controlled by acetaminophen. Transient fatigue(<8 hours) was reported by 25% of patients (with one grade 3),and pruritus (16% of patients) and edema (24% of patients)were also fairly common. These symptoms were usually mildand of short duration. A summary of frequent adverse events ispresented in Supplementary Fig. S1. Adverse events that wereobserved in <10% of patients but that were at least possiblyrelated to the treatment included chills, headache, pyrosis, anddry oral mucosa. One patient presented with an enlarged groinLN, and one had eczema. No adverse effect recorded wasconsidered serious.

Clinical responses to L19–IL2 treatmentAccording to the modified irRC, 6 of 24 evaluable patients

(25%) presented an overall irCR at the end of treatment.Twenty-five percent of patients presented overall irPR, whilethe disease remained stable in another 29.2% of patients(irSD). Finally, 20.8% of patients showed a PD (irPD).Detailed overall tumor response in each patient accordingto both modified irRC and RECIST is summarized in Sup-plementary Table S1.

We also evaluated the response at the level of individualmetastases. In total, 514 separately treated metastases, (63index lesions and 451 nonindex lesions) could be evaluatedfor local tumor response (Table 2). Analysis of the results

Table 1. Patients' characteristics

CharacteristicNumber ofpatients (n ¼ 25) Percent

SexMen 14 56Women 11 44

StageIIIB 4 16IIIC 21 84

Site of treated metastasesCutaneous 14 56Subcutaneous 8 32Both 3 12

Total number of treated metastases<20 17 68�20 8 32

Previous therapiesSurgery 25 100Limb perfusion 4 16Radiotherapy 4 16Adjuvant IFNa 12 48Systemic chemotherapy 3 12Intralesional IL2 4 16

Table 2. Locoregional tumor response (modified irRC)

Response, index lesions Response, nonindex lesions

Response N (%) N (%)

irCR 28 (44.4) CR 205 (45.4)irPR 6 (9.5) Non-CR 246 (54.6)irSD 23 (36.5)irPD 6 (9.5)

Total 63 (99.9) 451 (100)

irCR, complete response (disappearance of any evidence of vital tumor).irPR, partial response (a�50% decrease in the product of the two largest perpendicular diameters (LPD) of the lesion compared withbaseline).irSD, stable disease (a variation in LPDs, which was neither sufficient to qualify the lesion as irPR nor as irPD).irPD, progressive disease (a �25% decrease in the product of LPDs of the lesion compared with baseline).CR, complete response (disappearance of any evidence of vital tumor).Non-CR, non-complete response.






revealed that 53.9% of all index lesions exhibited an objec-tive response (44.4% irCR and 9.5% irPR), 36.5% of indexlesions remained stable, and 9.5% of index lesions pro-gressed. When the analysis was extended to nonindexlesions (n ¼ 451), a CR (disappearance) was observed in205 lesions (45.4%), while another 246 lesions showed a non-CR (54.6%).

Figure 1 illustrates the clinical course of 5 patients withcomplete responses (Fig. 1A–C), partial responses (Fig. 1D), orSD (Fig. 1E). Further details are reported in the SupplementaryData.

Progression-free and overall survivalToevaluate the progression andOS of this cohort of patients,

data were collected during the study and beyond the 12-monthfollow-up period.

In 20 of 24 evaluable patients, disease progressed after thestart of L19–IL2 treatment. The first recurrence site was

locoregional (new cutaneous/subcutaneous metastases) in all20 patients, who progressed further in the course ofdisease. Figure 2A shows Kaplan–Meier curves reporting theprogression-free survival (PFS) data for progression to newcutaneous/subcutaneous metastases (skin), LN metastases, ordistant metastases (systemic), respectively.

In 19 of 24 patients, a locoregional recurrence to eithercutaneous/subcutaneous metastases or LN metastases wasobserved within 1 year from the first treatment. The mediantime to locoregional progression was 93 days (range, 41–864days). However, in 5 patients, no further lesion appeared for 2years or longer.

Among the 19 patients who showed recurrence tolocoregional sites (cutaneous/subcutaneous and LN), 9patients progressed further to systemic disease. The medianprogression time to systemic disease was 331 days (range, 67–960 days). However, 15 patients remained free of distantmetastases for time intervals ranging from 130 to 1,015 days.

Figure 2B shows the OS data for the 24 evaluable patients.Three patients died, with one each at 278, 616, and 966 daysafter the first treatment. The other 21 patients had survived atthe timepoint of the last follow-up for periods ranging from130to 1,048 days. The median survival time is 905 days.

Analysis of systemic immune responsesAltogether, 45 blood samples from17 patientswere available

for the evaluation of systemic immune responses. Completeresults are presented in Table 3. A significant increase in thefrequency of CD4þCD25þFox-P3þ Tregs was observed in 15 of16 evaluable patients after 4weeks of treatment comparedwithbaseline (mean frequency, 12.1% vs. 8.5%; P < 0.001). Afterstopping treatment, frequencies returned to baseline values atday 85 (Fig. 3A). There was a decrease of CD14þCD11bþHLA-DR�/low MDSCs from 11.7% at baseline to 8.7% at day 29 and7.6% at day 85 (Fig. 3B). Both the differences between baselineand end of treatment and between baseline and day 85 werestatistically significant with P values of 0.001 and 0.002, respec-tively. No change in frequency was observed for NK, CD4þ, orCD8þ T cells. In contrast with the frequency, the absolutenumbers of NK cells (P¼ 0.005; Fig. 3C) and CD8þ T cells (P¼0.007) were increased at day 29 and decreased thereafter tobaseline values. The absolute lymphocyte count increased untilday 29, mainly driven by an increase of CD4þ T cells, andremained elevated several weeks after day 85 as comparedwithbaseline (P ¼ 0.009; Fig. 3D).

The proportions of patients with detectable antigen-specificT cells were 47.1% at baseline, 29.4% at day 29, and 27.3% at day85 for Melan-A–specific T cells and 52.9%, 58.8%, and 45.5% forNY-ESO-1–specific T cells. None of these changes was statis-tically significant. The de novo appearance of specific T cellsbetween baseline and day 29 was observed in 3 patients forMelan-A– and 2 for NY-ESO-1–specific T cells, but converselysome patients also lost a preexistent baseline T-cell reactivityat day 29 (6 patients forMelan-A– and 1 for NY-ESO-1–specificT cells). No change in reactivity was observed among theremaining patients.

Changes in the frequency of NY-ESO-1–, Melan-A–, andinfluenza-specific T cells upon treatment were analyzed

Figure1. Clinical findings in patientswho exhibited complete (A, B, andC),partial (D), or SD (E) responses. Scale bars, 10 mm.

Weide et al.





separately for CD8þ and CD4þ T cells and for each of sixcytokines (Supplementary Table S2). A significant decreaseduring therapywas observed for the proportion of IFN gþCD4þ

T cells. A nonsignificant tendency was observed for a dec-rease in the frequency of IFNgþCD8þ, TNFaþCD8þ, andTNFaþCD4þ T cells (all P > 0.100) upon stimulation withNY-ESO-1 peptides.Upon stimulationwithMelan-A peptides, we again observed

a tendency for decreasing levels of IFNgþCD4þ andTNFaþCD4þ T cells (P > 0.100) and no change in the CD8þ

Melan-A–specific cells.All changes were temporary, as no significant differences in

the frequency of antigen-specific T cells were observed in acomparison of baseline and day 85. No changes in the fre-quency of influenza-specific T cells were observed between thedifferent time points.No significant predictive or surrogate markers for clinical

response could be identified among all the analyzed immune-cell parameters.

DiscussionTo the best of our knowledge, this is thefirst study to address

the use of an IL2-based immunocytokine for the intralesionaltreatment of satellite or in-transit metastases in patients withmelanoma. The treatment was generally well tolerated, withfew adverse events, limited in grade and similar in nature tothose recorded for untargeted IL2.

In terms of efficacy, the objective response rate recorded inthis study was lower than that shown by untargeted IL2. Thisdifference deserves some comment.

In previous studies using untargeted IL2, we reported CRs ofinjected metastases, according to adapted RECIST criteria, inmore than 60% of patients (14, 15), which is significantly higherthan the CR rate of 25% according to modified irRC/RECISTobserved here. In our opinion, this difference does not reflect alower efficacy of L19–IL2 compared with untargeted IL2, but ismore likely related to differences in the patient populationsand trial designs. First, the two studies that used untargetedIL2 were carried out in patients whose disease stage was evenlydistributed among stages IIIB, IIIC, and IV (24, 25, and 23patients, respectively). The results of the follow-up study (32)clearly show that patients who benefited the most, in terms ofOS, were those in stage IIIB at the screening visit (86.8% 5-yearsurvival rate), as opposed to patients in stage IIIC, who onlyshowed a 31.4% 5-year survival rate. This last value was close tothat recorded for stage IV M1a patients (16.7% 5-year survivalrate). In the present study, most patients (21 of 25) were instage IIIC at screening and only 4 were at stage IIIB. This mightexplain the lower percentage of patients who, in our study,could be classified as CR at the EoS visit. Second, there aredifferences between the studies with untargeted IL2 and thepresent study in the treatment regimen and schedule. In IL2-based studies, the treatment schedule was two to three timesweekly with a maximum daily dose of 16 MIU IL2 or variable,according to the individual tumor burden of patients (14, 15).

Figure 2. Progression and survivalanalysis. A, Kaplan–Meier curvesshowing PFS time intervalsmeasured in patients for whom thediseaseprogressed to locoregionalcutaneous/subcutaneousmetastases (skin), or locoregionalLN, or to distant metastases(systemic). For the calculation ofLN and systemic, priorlocoregional cutaneous/subcutaneous metastases wereignored. Censored events areindicated by a vertical tick.B, Kaplan–Meier analysis of OS in24 evaluable patients.






Importantly, in these trials, new lesions arising during treat-ment were also injected, and treatment continued until alllesions, including the new ones, finally regressed. Therefore,the overall length of treatment varied between 1 and 57weeks (median, 7.5 weeks). In the present study, L19–IL2 wasintratumorally delivered once per week for 4 consecutiveweeks only and at the constant maximum daily dose of 10MIU, evenly distributed among all injectable lesions. Newlesions were not injected.

We are aware of both advantages and limitations inherentto the choice of a highly standardized intratumoral deliverymodality for patients in whom the tumor burden differs greatly(i.e., variable amount of injected drug per lesion, possiblysuboptimal treatment duration). The rationale behind thischoice was that (i) the intralesional approach would allowreaching higher concentrations of the drug in the lesion (ascompared with systemic delivery), as we assume that very highIL2 concentrations are required for efficacy; and (ii) theantibody would ensure a longer residence time of L19–IL2 inthe lesions, therefore allowing a reduced number of injectionsas compared with IL2. A standardized schedule with fewerinjections and shorter treatment duration would reduce injec-tion pain and discomfort for patients and facilitate logistics.

The rate of objective responses recorded in the two sets ofstudies is of comparable magnitude in any case, with differ-ences possibly attributable to different distribution of patientsamong the disease stages.

An analysis of PFS for the patients enrolled in the presentstudy shows that, although most patients relapsed within 1year to new in-transit/satellite metastases, progression toproximal LN and to distal systemic disease occurred in arelatively low number of patients and was much slower. In2010, Romano and colleagues (33) published a retrospectivestudy based on clinical records of patients with stage IIImelanomawith no evidence of disease (NED) seen atMemorialSloan-Kettering Cancer Center (New York, NY) between 1992and 2004, who ultimately relapsed to locoregional, nodal, orsystemic disease. Among relapsing patients, 340 had adequatefollow-up to be evaluable for all parameters. In that study,similar to our study, most NED stage IIIC patients (�80%) whorelapsed to locoregional disease did so in less than 1 year.However, the data also show that progression to nodal diseaseof NED stage IIIC patients occurred even faster (90%of patientswho progressed did so after 1 year), and that progression tosystemic disease was observed in virtually all patients who hadrelapsed after 2 years of follow-up.

Table 3. Analysis of systemic immune responses

Cell subsetDefinition/phenotype Reference

Baseline(mean)N ¼ 16

End oftreatment(mean)N ¼ 17

Follow-up(mean)N ¼ 11

Absolute WBC count Blood count mL 5,901 7,009d 6,438Absolute lymphocyte count Blood count mL 1,476 2,005a 1,937c

Frequency lymphocytes Blood count WBC 25.3% 29.0%d 30.6%c

Absolute CD4þ lymphocytes CD4þ mL 767 1,040a 1,040d

Frequency CD4þ lymphocytes CD4þ Lymphocytes 51.7% 52.8% 53.5%Absolute CD8þ lymphocytes CD8þ mL 293 398c 397Frequency CD8þ lymphocytes CD8þ Lymphocytes 19.8% 19.3% 20.2%Absolute NK cells CD16þCD56low mL 106 166c 137Frequency NK cells CD16þCD56low Lymphocytes 7.4% 8.2% 7.4%Tregs CD25þFoxP3þ CD4þ T cells 8.5% 12.1%a 8.3%b

MDSCs CD14þCD11bþ HLA-DR�/low PBMCs 11.7% 8.7%c 7.6%c

Cell subsetDefinition/phenotype Reference

Baseline,% N ¼ 17

End oftreatment,% N ¼ 17

Follow-up,% N ¼ 11

Melan-A–specific T cells Detectable No. of analyzedpatients

47.1% 29.4% 27.3%

NY-ESO-1–specific T cells Detectable No. of analyzedpatients

52.9% 58.8% 45.5%

NOTE: Statistically significantly changed values are highlighted by bold letters.Abbreviation: WBC, white blood cells.aP < 0.001 compared with baseline.bP < 0.001 compared with end of treatment.cP < 0.01 compared with baseline.dP < 0.05 compared with baseline.

Weide et al.





Our study indicates that only 17% (4 of 24) and 25% (6 of 24)of patients had progressed to nodal or systemic disease at 1 or 2years after the first intralesional treatment with L19–IL2,respectively. Although obtained in a much smaller cohort ofpatients, our data suggest that the control of in-transit/satellitemetastases, obtained through intralesional injection of L19–IL2, translates into a slower diffusion rate of the disease todistant sites.In terms of OS, 3 of the 24 patients died during the follow-

up period at 278, 616, and 966 days after first treatment,respectively. Our data indicate that 48% of patients survived>2 years from the date of first treatment, with another 44%having been monitored only for shorter times. Although wefind these data encouraging, it is still too early to compareour statistics with historical data (3), which show that 3-yearsurvival for patients with stage IIIC melanoma is approxi-mately 50%.Translational side studies in 17 patients revealed that intra-

tumoral treatment resulted in significant frequency changes ofimmune-cell populations in the peripheral blood. Besides atemporary increase in Tregs during treatment, observed inalmost all patients, we observed a sustained decrease ofMDSCs. These cells have been described to possess clinically

relevant immunosuppressive functions, at least in malignantmelanoma (28). Filipazzi and colleagues analyzed patientswithstage II/III melanoma with NED and found a trend for betterdisease-free survival in patients with low amounts of CD14þ

HLA-DR�/low MDSCs compared with those with a high fre-quency of these cells (P ¼ 0.08), but their patient cohort (n ¼33) was very small (34). Walter and colleagues reported astrong association of this particular MDSC population withnegative outcome and survival in patients with renal cellcancer after multipeptide vaccination (27). The decrease ofMDSC frequency might, therefore, partially explain the bene-ficial mode of action of intratumoral L19–IL2, represented bywhat seems to be a favorable DMFS rate as compared with thatfor historical controls. On the other hand, a transient increaseof Tregs was observed after IL2 therapies (35, 36). A directinteraction between IL2 used in a therapeutic intention andTregs can be assumed because of the high expression levels ofthe a-chain of the IL2 receptor (CD25) on Tregs. Nevertheless,the clinical impact and the functional state of these treatment-induced Tregs and of circulating Tregs in patients with mel-anoma in general remain unclear (reviewed in ref. 37). Incontrast withMDSCs, no prognostic role of Tregswas observedin what is thus far the largest study of patients with advanced

Figure 3. Systemic immuneresponses. A, frequency of CD4þ

CD25þFox-P3þ Tregs increasedin 15 of 16 evaluable patients.A significant increase in thefrequency of CD4þCD25þFox-P3þ

Tregs was observed after 4 weeksof treatment compared withbaseline (mean frequency, 12.1%vs. 8.5%;P < 0.001). After stoppingtreatment, frequencies returnedto baseline values at day 85. B,decrease of CD14þCD11bþHLA-DR�/low MDSCs starting from11.7% at baseline to over 8.7% atday 28 and to 7.6% at day 85.Differences between baseline andend of treatment and betweenbaseline and day 85 werestatistically significant (P ¼ 0.001and 0.002, respectively). C, anincrease in absolute numbers orCD16þCD56low NK cells wasobserved on day 29 (P ¼ 0.005),which might be explained by thesustained increase in absolutelymphocytes upon treatment (D).






melanoma published recently (38). Moreover, a sustainedincrease in the frequency and absolute count of lymphocytes(mainly of the CD4þ T cells) and the temporary increase ofNK cell count was observed here, as previously reported forIL2-based treatments (20). The analysis of NY-ESO-1– andMelan-A–specific T cells, for which a strong prognosticimpact was previously demonstrated by our group in stageIV patients (31), was inconclusive in this trial. At day 29,fewer patients had detectable Melan-A–specific T cells com-pared with baseline, with a tendency to decrease in frequen-cy for these cells. Frequency and detection rate returned tolevels similar to baseline after the treatment was stopped.These observations have to be interpreted with cautionbecause they are based on a small sample size and werenot confirmed by statistical testing. A significant decrease ofthe CD4þIFNgþ T-cell level was observed after stimulationwith NY-ESO-1 peptides in a comparison of day 29 withbaseline, but, in contrast, NY-ESO-1–specific T cells weredetectable in general in one more patient at day 29. Even if adecrease of melanoma-specific T-cell responses upon L19–IL2 treatment was postulated (which cannot be demonstrat-ed from data presented here), the biologic significance ofsuch a potential temporary decrease in the peripheral bloodwould remain unclear. The decrease in the frequency of NY-ESO-1– or Melan-A–specific CD4þ cells can be partly attrib-uted to the significant absolute increase of CD4þ cell counts.Moreover, it might be a correlate of T-cell trafficking fromthe circulation to metastatic tissue in the periphery duringactive therapy and would be in agreement with the obser-vation that tumor sites become strongly infiltrated byimmune cells before rejection.

The assessment of response to intralesional treatment inmelanoma is very challenging, as it is not always possible todecide whether a lesion has completely disappeared or not.This is true for both cutaneous lesions, which after treatmentmay leave behind hyperpigmented regions of difficult classi-fication (see, e.g., Fig. 2E), and subcutaneous lesions, as sonog-raphy or CT may still generate abnormal signals in lesionsdevoid of tumor cells (39), where heavy lymphocytic infiltrationhas occurred or necrotic/fibrotic areas have developed. Thissuggests that the existing response evaluation guidelines arenot optimal and raises the need for the scientific community toconsider new, ad hoc evaluation criteria and study endpointsfor this pathology and treatment modality.

In this study, in addition to evaluating objective response,we also analyzed response separately for each individualinjected lesion present at baseline. New lesions were neitherinjected in the trial nor evaluated in this particular assess-ment. At EoS, tumor burden of index/target lesions wascompared with that present at baseline and used to classifythe patient as CR, PR, SD, or PD. Response of nonindex/nontarget lesions, which were almost exclusively smallerthan 5 mm in size and therefore difficult to assess withregard to the exact size, was only classified as either CR(complete disappearance), which was confirmed by histo-pathology in equivocal cases, or as non-CR at day 85 and EoS.We believe that, even in the presence of the describeddifficulties in unambiguously assessing responses, this addi-

tional analysis limited to injected baseline lesions gives afair representation of the efficacy of L19–IL2 for intrale-sional treatment of patients with stage III melanoma.However, it seems that DMFS (i.e., time to stage IV) is amore solid and clinically meaningful endpoint for compar-ing long-term efficacy of immunocytokines or other biolo-gics in intralesional applications to surgery or systemictreatments.

It seems reasonable to allow the treatment of all injectablelesions, including new metastases developing after the startof therapy over a defined interval of time, and to assess theduration of benefit for the patient in terms of both theabsence of local progression and "time to stage IV." A longperiod with local control of disease and absence of distantmetastasis implies that patients will not have to undergotoxic treatments for metastatic disease, which may acceler-ate the emergence of mutations and resistant cancer cells(40–45).

Recently, in a preclinical study, Schwager and colleagues (46)have shown that L19–IL2 is able to induce complete tumorremissions in 100% of treated mice when administered in asingle intratumoral injection in combination with L19–TNF.Interestingly, the two agents did not lead to cures whenadministered as single agents. The effect could be obtainedonly in immunocompetent mice (but not in nude mice),indicating the requirement of T cells for the complete erad-ication of tumors. Both of these therapeutic agents have beenextensively studied individually in clinical trials (20, 21, 47, 48),and the antigen recognized by the L19 antibody has anidentical sequence in mice and humans. These findings pro-vided a rationale for a combination trial inmelanoma, which iscurrently ongoing.

In conclusion, intratumoral L19–IL2 treatment elicitedobjective responses in a high percentage of lesions. Thetargeted form of IL2 yielded results similar to those of theuntargeted cytokine, but at lower cumulative doses and witha more favorable schedule and treatment duration. Inintratumorally treated patients, we observed a sustaineddecrease of circulating immunosuppressive MDSCs andpromising data regarding the time to distant progressionas well as OS.

Disclosure of Potential Conflicts of InterestB. Weide is a consultant/advisory board member for Philogen. T.K.

Eigentler is a consultant/advisory board member for Bristol-Myers Squibb.D. Neri has an ownership interest (including patents) in Philogen S.p.A. and isa consultant/advisory board member for the same. R. Gutzmer has receivedother commercial research support and project support from Roche, Johnson& Johnson, Novartis, and Pfizer, has received honoraria from the speakersbureaus of Bristol-Myers Squibb, Roche, GlaxoSmithKline, Pfizer, Janssen,Merck Serono, MSD/Merck, and Amgen, and is a consultant/advisory boardmember for Bristol-Myers Squibb, GlaxoSmithKline, Novartis, Roche, andMSD. C. Garbe has received commercial research support from Bristol-MyersSquibb, GlaxoSmithKline, Roche, and SOBI and is a consultant/advisoryboard member for Bristol-Myers Squibb, GlaxoSmithKline, MSD, PhilogenS.p.A., Roche, and SOBI. No potential conflicts of interest were disclosed bythe other authors.

Authors' ContributionsConception and design: B. Weide, D. Neri, C. GarbeDevelopment of methodology: B. Weide, H. Zelba, D. Neri

Weide et al.





Acquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): B. Weide, T.K. Eigentler, A. Pflugfelder, H. Zelba,A. Martens, L. Giovannoni, R. Gutzmer, J.C. Becker, C. GarbeAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): B. Weide, T.K. Eigentler, H. Zelba, G. Pawelec,L. Giovannoni, P.A. Ruffini, G. Elia, D. Neri, J.C. Becker, C. GarbeWriting, review, and/or revision of themanuscript:B.Weide, T.K. Eigentler,A. Pflugfelder, H. Zelba, A. Martens, G. Pawelec, L. Giovannoni, P.A. Ruffini,G. Elia, D. Neri, R. Gutzmer, J.C. Becker, C. GarbeAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): A. Martens, G. Pawelec, L. Giovannoni,G. EliaStudy supervision: B. Weide, P.A. Ruffini, D. Neri, J.C. Becker, C. Garbe

Grant SupportThis study was supported by the EU Seventh Framework Programme "PRIAT"

(Profiling Responders In Antibody Therapies), grant agreement no 305309.Philogen S.p.A. financed the article-processing charge.

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicate thisfact.

Received November 22, 2013; revisedMarch 18, 2014; acceptedMarch 18, 2014;published OnlineFirst April 4, 2014.

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Weide et al.




2014;2:668-678. Published OnlineFirst April 4, 2014.Cancer Immunol Res Benjamin Weide, Thomas K. Eigentler, Annette Pflugfelder, et al. Immunologic Responses

IL2 Results in Sustained Clinical and Systemic−with L19 Intralesional Treatment of Stage III Metastatic Melanoma Patients

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