Combined treatment with ipilimumab and intratumoral interleukin-2 in pretreated patients with stage IV melanoma—safety and efficacy in a phase II study

Abstract

Treatment of advanced melanoma patients with ipilimumab results in improved survival. However, only about 20% of treated patients experience long-term benefit. Combining treatment of ipilimumab with other drugs may improve immune activation and potentially enhance clinical efficacy. The aims of the phase II clinical trial reported here were to investigate tolerability and efficacy of a combined immunotherapeutic strategy comprising standard systemic ipilimumab at 3 mg/kg four times at 3-week intervals and intratumorally injected IL-2 at 9 MIU daily twice weekly for four weeks in pretreated melanoma patients with distant metastasis. The primary endpoint was the disease control rate according to immune-related response criteria at week 12; tolerability according to Common Terminology Criteria for Adverse Events criteria was secondary endpoint. No objective responses were observed in the 15 enrolled patients. Three patients had stable disease 12 weeks after starting treatment, yielding a disease control rate of 20%. Tolerability of this combination treatment was acceptable. Observed adverse events were those expected from the respective monotherapies. Autoimmune colitis was observed in two patients. Grade III/IV adverse events were observed in 40% of patients, and no treatment-related deaths occurred. Thus, this combined immunotherapy is associated with adverse events similar to those associated with the respective monotherapies. However, this study does not provide any evidence of improved efficacy of the combination over ipilimumab alone.

Electronic supplementary material

The online version of this article (doi:10.1007/s00262-016-1944-0) contains supplementary material, which is available to authorized users.

Introduction

Intratumoral application of drugs is an appealing therapeutic concept, as high concentrations of a drug can be directly delivered to the tumor, while systemic concentrations remain low. Thus, this strategy is particularly promising if both—efficacy and toxicity of an agent—are increasing in a dose-dependent manner. Systemic treatment with IL-2 can result in durable clinical responses. However, benefit is limited to a rather small proportion of melanoma patients and treatment at barely tolerated doses is required [1]. Lower systemic doses of IL-2 were ineffective [2, 3]. IL-2 is known as a non-specific T cell activator based on the observation of a strong IL-2-dose-dependent lympho-proliferation in vitro. However, because a high proportion of T cells in the tumor microenvironment are assumed tumor specific, local application of IL-2 may preferentially activate melanoma-specific responses accordingly. A strong local inflammatory reaction appearing within 1–2 weeks, the histopathological observation of a strong T cell infiltrate in regressing lesions [4] and of vitiligo-like local depigmentation [5] are in agreement with this assumed mode of action.

The intratumoral delivery of IL-2 was initially tested with the intention to achieve higher local responses of the injected lesions (due to higher local concentrations) but lower systemic side effects (due to a lower total dose) compared to the high-dose systemic treatment with IL-2. In clinical trials, we found that repeated intratumoral injections of IL-2 into melanoma metastases represent a highly efficient local treatment particularly for patients with multiple small cutaneous lesions [4, 5]. Thus, intratumoral IL-2 represents a potentially curative alternative to surgery or systemic treatments for a subset of patients.

In addition to its local efficacy, based on preclinical findings in animal models, a beneficial systemic effect may also accrue [6, 7]. Maas et al. reported the regression of distant non-injected tumors after direct treatment in lymphoma-bearing mice. Systemic treatment with the same IL-2 doses was far less effective [6]. Similarly, van Es et al. used a rabbit carcinoma model and reported regression of non-injected tumors. Interestingly, a second challenge of cured animals with tumor cells was not possible, suggesting the generation of specific immunity [7]. Local proliferation followed by systemic dissemination of activated T cells may serve as an explanation. Alternatively, the distant effect may be the consequence of direct or indirect activation of antigen-presenting cells in the tumor microenvironment upon local IL-2 treatment. After antigen uptake, these cells may subsequently migrate to the lymph nodes and initiate immune responses, and are thereby contributing to a locally induced, but systemically active in situ vaccination effect. Clinically, regression of non-injected lesions and a good long-term outcome has been observed in patients after IL-2-based intratumoral treatments [8, 9]. An increase in the frequency of T cells targeting melanoma-associated antigens [5] and a decrease of MDSCs in the peripheral blood during treatment are also in agreement with a potential systemic effect. Systemic treatment with ipilimumab, an antagonistic monoclonal human IgG1 antibody binding CTLA-4, demonstrated improved OS in metastatic melanoma [10, 11]. However, long-term survival is limited to approximately 20% of patients [12]. The exact mode of action of ipilimumab has also not been fully elucidated. CTLA-4 is expressed on CD4⁺ and CD8⁺ T cells after initial activation [13] as well as constitutively on regulatory T cells (Tregs) [14]. It is a key element in tolerance regulation and competes with a higher affinity than CD28 for binding to the same ligands B7.1 (CD80) and B7.2 (CD86) on antigen-presenting cells [15–17]. CTLA-4 engagement induces T cell tolerance and anergy without the induction of cell death [18, 19]. Physiologically, these interactions contribute to the maintenance of the delicate equilibrium between T cell reactivity against foreign antigens but tolerance of self-epitopes and normal tissue protection [18]. A direct mode of action is most likely enhanced anti-tumor functions of T cells induced through the blockade of expressed CLTA-4 [20]. A delayed increase in both circulating CD4⁺ and CD8⁺ T cell frequencies correlated with the patients’ OS and response after the first dose of ipilimumab [21]. Regarding the specific targets of T cells, several recent studies highlight the role of T cell responses targeting neo-epitopes for a favorable clinical response to ipilimumab [22–24] or nivolumab [25]. Whether ipilimumab amplifies preexisting immune responses [26] or rather broadens the T cell target spectrum [22] or both remains a matter of debate.

The rationale for the combined treatment tested here was a potential synergistic immunologic effect of ipilimumab and intratumoral IL-2 primarily in the microenvironment regarding the induction/amplification of T cell responses. Indeed, one study performed in mice revealed an important role of high IL-2 concentrations for anti-tumor activity of CTLA-4 blockade [28]. Moreover, the potential systemic effects mediated by melanoma-specific T cells after intratumoral IL-2 may be secondarily amplified by ipilimumab. Thus, the aims of the current study were to investigate whether the combination of ipilimumab with intratumoral IL-2 is tolerated and demonstrates efficacy in pretreated patients with metastatic stage IV melanoma.

Materials and methods

Study design

This was a prospective open-label, single arm, phase II clinical trial (NCT01480323). Sample size was calculated according to a Simon two-stage design with n = 15 evaluable patients required for part one and a total sample size of n = 41 in case of proceeding to part two. The study was stopped after part one as protocol-defined efficacy criteria (stable disease (SD), partial response (PR), or complete response (CR) in at least 6 of 15 patients) were not met according to a pre-planned interim analysis. The primary endpoint was the disease control rate according to immune-related response criteria (irRC) at week 12 (irDCR). Secondary endpoints were tolerability according to Common Terminology Criteria for Adverse Events criteria (version 4), overall survival 12 months after first dosing, overall response rate after 12 weeks and best overall response rate according to irRC and modified World Health Organization (mWHO) criteria.

Patients

The study was approved by the Ethics Committee Tübingen (506/2011AMG1), and patients were included after written informed consent. Inclusion criteria were histologically confirmed melanoma, stage IV according to the American Joint Committee on Cancer (AJCC) 2009 classification [27], Eastern Cooperative Oncology Group performance status ≤1, life expectancy >3 months, age ≥18 years, no persisting acute toxicity associated with prior therapy, measurable disease with the presence of at least one injectable lesion >5 mm (longest diameter) or at least 5 injectable lesions <5 mm. Patients must have received at least one line of prior systemic therapy for metastatic disease with relapse following a clinical response, or failure to demonstrate a clinical response, or failure to tolerate such a regimen due to toxicity. Exclusion criteria were pregnancy or breast-feeding women and patients with any other malignancy from which the patient had been disease free for less than 5 years, with the exception of adequately treated and cured basal or squamous cell skin cancer, superficial bladder cancer or carcinoma in situ of the cervix. Other exclusion criteria were presence of active infections, autoimmune disease, untreated symptomatic central nervous system metastases and concurrent medical conditions requiring the use of systemic steroids.

Treatments and assessments

IL-2 (Proleukin^®) was given by intratumoral injection of 9 million international units (MIU) per day on days 1, 4, 8, 12, 15, 19, 22 and 26. The administered dose was distributed between all injectable soft tissue metastases (minimum of 1 and up to a maximum of 5 injected lesions). Injections were guided by sonography for deep soft tissue metastases. Ipilimumab was infused at a dose of 3 mg/kg, on days 2, 23, 44 and 65.

Response evaluation was primarily performed according to irRC [28]. CR, PR and progressive disease at week 12 had to be confirmed after 4–6 weeks for evaluation of the primary endpoint. Additionally, response evaluation was performed according to mWHO criteria. Radiologic whole-body tumor assessments by computed tomography or by MRI were performed at baseline and every 12 weeks thereafter in the absence of confirmed progressive disease until week 48.

Translational side studies

Peripheral blood samples were derived from blood draws taken within 28 days before the first dose of IL-2 (baseline), after 4 weeks (early time point; days 26–33), or 12 weeks after treatment start at the time of the tumor assessment (late time point; days 77–96). PBMCs were isolated using standard Ficoll/Hypaque density centrifugation, viably cryopreserved and stored in liquid nitrogen until usage. Freshly thawed PBMCs were immediately analyzed by flow cytometry. In all antibody panels, Fc receptors were blocked with human IgG (Gamunex; Talecris, USA), and dead cells were excluded by ethidium monoazide labeling (Biotinum, USA). Staining was performed separately for the analysis of MDSCs (panel 1) and Tregs (panel 2). After surface staining but prior to intracellular antibody staining, cells for determination of Tregs were fixed and permeabilized using the Human FoxP3 Buffer Set (BD Pharmingen, USA). Details of respective antibody panels are described in Supplementary Table 1. Data were acquired with a BD LSR-II with FACSDiva software V6.1.3 (BD Biosciences, USA) and analyzed with FlowJo V9.3.2 (Tree Star, USA). Detailed representative gating strategies are displayed in Supplementary Fig. 1.

Statistics

Survival probabilities were estimated using the Kaplan–Meier method and compared by log-rank testing. Death from any reason was considered as an “event,” whereas patients who were alive at the time of last follow-up were censored. Categorization of patients for the respective cell populations (Supplementary Table 2) of interest was performed using previously published cutoff points [29]. Changes in peripheral blood-derived routine clinical factors and immune parameters during treatment with IL-2 and ipilimumab were assessed using Wilcoxon matched pairs testing. Correlations between best overall response rate and proportions of cellular compartments were analyzed by Chi-square and Fisher’s exact tests (two-sided). p values <0.05 were considered significant in all analyses. SPSS22 (IBM) was used to perform all statistical analyses.

Results

Patients and treatments

Patients from the Department of Dermatology (Tübingen, Germany) were enrolled between February 2012 and June 2014. Patient characteristics are described in detail in Table 1. The median age at registration was 54.0 years. Nine of the 15 patients (60%) were male. All patients had stage IV melanoma. 40.0% of patients (n = 6) were M stage M1a, 13.3% (n = 3) M1b and 46.7% (n = 7) M1c.

Table 1.

Baseline characteristics

Factor	Category	N	%
Age	≤50 years	5	33.3
	>50 years	4	26.7
	>60 years	2	13.3
	>70 years	4	26.7
Gender	Male	9	60.0
	Female	6	40.0
M category	M1a	6	40.0
	M1b	2	13.3
	M1c	7	46.7
Prior systemic treatments for metastatic disease	BRAF inhibitor/MEK inhibitor	6	40.0
	Chemotherapy	11	73.3
Treatment line	Second line	15	100
	Third line	2	13.3
Presence of brain metastasis	Yes	0	0
	No	15	100
Subsequent treatments	None	8	53.3
	PD-1antibody/PD-L1antibody	2	13.3
	BRAF inhibitor/MEK inhibitor	4	26.7
	Chemotherapy	4	26.7
ECOG	0	14	6.7
	1	1	93.3
IL-2 doses	8	12	80.0
	7	2	13.3
	5	1	6.7
Ipilimumab doses	4	11	73.3
	3	1	6.7
	2	2	13.3
	1	1	6.7
LDH	Elevated	8	53.3
	Normal	7	46.7

ECOG eastern cooperative oncology group, IL-2 interleukin-2, LDH lactate dehydrogenase, PD-1 programmed death-1, PD-L1 programmed death ligand-1

The full course of ipilimumab was administered to 11 of the 15 patients (73.3%), while ipilimumab was stopped after three (n = 1), two (n = 2), or one dose (n = 1) in the remaining patients. 12 patients (80.0%) received the full course of IL-2 therapy with 9 MIU total dose applied twice weekly for 4 weeks. Two patients missed one dose of IL-2 treatment. For one patient, only a single dose of IL-2 was given. Two patients prematurely terminated study therapy due to rapid tumor progression and one because of an adverse event/toxicity. In total, 10 of 15 patients received all 8 doses of IL-2 and all 4 doses of ipilimumab.

Tolerability

All 15 patients were evaluable for tolerability. Table 2 lists adverse events which were observed in at least two patients (>10% of all patients). Adverse events which were unlikely to be, possibly or probably related to either IL-2 or ipilimumab according to the investigator´s judgement, were considered. Localized grade I/II injection site reactions (pain, edema, erythema) were observed in all patients. Fatigue or flu-like symptoms appeared usually within the first 24 h after IL-2 injections, both being observed in 60% of patients. In 2/9 patients with fatigue, grade III severity was reported. Colitis and exanthema, judged as immune-related (ir) adverse events, were observed in 13 and 40% of patients, respectively. Colitis was seen at grade III in one patient. In addition, a grade IV increase in γ-glutamyl transferase and grade III increase in the absolute neutrophil counts were observed in one patient who experienced major disease worsening over this time period and died three weeks later from melanoma progression. Thus, it was unlikely that this was related to the study treatments.

Table 2.

Adverse events

Adverse event	Any grade		Grade III/IV
	N	%	N	%
Any	15	100	6	40
Injection site reaction including injection pain	15	100	0	0
Fatigue	9	60	2	13
Flu-like symptoms	9	60	0	0
Pain (excluding injection pain)	7	47	2	13
Rash	6	40	0	0
Nausea	5	33	0	0
Anorexia	4	27	0	0
Constipation	3	20	0	0
Pruritus	3	20	0	0
Anemia	2	13	0	0
Colitis	2	13	1	7
Dyspnea	2	13	0	0
Hyperhidrosis	2	13	0	0
Insomnia	2	13	0	0

The following serious adverse events were observed during the trial: Patient 1 was hospitalized due to increasing grade II malaise, fatigue and decrease in the general condition after he had received the full course of IL-2 therapy and 3 cycles of ipilimumab. He was already under poor conditions at start of treatment (Eastern Cooperative Oncology Group performance status 1) with a serum LDH 2.5-fold above the upper limit of normal and multiple metastases located to his liver, bones, soft tissues and lymph nodes. This patient had not responded to any of the previous systemic treatments including chemotherapy and targeted therapy. Computed tomography scans revealed marked disease progression with new ascites and pleural effusions at the time point of hospitalization. The patient died within a few days from melanoma progression. Patient No. 5 was hospitalized with grade 3 pain of the back. Radiologic assessments proved a new pathologic fracture in the vertebral column. The pain improved after symptomatic therapy with analgesics. Patient No. 13 was hospitalized because of grade III diarrhea/colitis. A colonoscopy including biopsies confirmed the diagnosis of autoimmune colitis, which recovered after symptomatic therapy and immunosuppressive treatment with intravenous prednisolone. The same patient subsequently developed sepsis related to a venous port infection. She died despite surgical port removal and antibiotic treatments two weeks after onset of sepsis. No causal relationship with the study treatments was reported. Patient No. 14 was hospitalized because of increasing serum creatinine and abdominal pain grade III. Radiologic assessments showed increasing renal melanoma metastasis. Renal insufficiency due to tumor infiltration in combination with a urinary infection was diagnosed. Study treatments were terminated, and treatment with a BRAF inhibitor was initiated.

Efficacy

The primary endpoint of this study was the irDCR at week 12. All 15 patients were evaluable for the primary endpoint. Three of the 15 patients (20.0%) showed SD at week 12 and 10 (66.7%) showed progressive disease at week 12 (Table 3). For 2 of the 15 patients (13.3%), the end of treatment visit including tumor assessment was performed before week 12 due to progressive disease requiring a different systemic treatment. Therefore, the irDCR at week 12 amounts to 20.0% (CI 95%: 4.33–48.09). Thus, the protocol-defined criteria for recruitment of additional patients in part 2 according to the Simon two-step design (irDCR ≥ 40%) were not met. The irDCR at week 12 was also 20.0% in the sub-analysis of 10 patients who received the complete treatments (4 doses of ipilimumab and 8 doses of IL-2). Objective responses were neither observed until week 12 nor thereafter in those patients with SD at week 12. Thus, the overall response rate at week 12 as well as the best overall response rate according to irRC and mWHO criteria was 0%. During follow-up, 12 patients died, while 3 were alive at the time of last observation. The reason of death was melanoma progression for 11 and sepsis for one patient. Median follow-up time was 231 days (188 days for patients who died and 940 days for those who were alive). The OS survival probability according to Kaplan–Meier was 33.3% after 1 year and 26.7% after 2 years in the entire cohort (Fig. 1).

Table 3.

Responses according to irRC and mWHO criteria at week 12

	Clinical response according to irRCa at week 12		Clinical response according to mWHO criteriab at week 12
	N	%	N	%
Complete response	0	0.0	0	0.0
Partial response	0	0.0	0	0.0
Stable disease	3	20.0	1	6.7
Progressive disease	10	66.7	12	80.0
Missingc	2	13.3	2	13.3
Total	15	100	15	100

^aImmune-related response criteria

^bModified World Health Organization criteria

^cDeath due to melanoma progression without radiologic tumor assessment before death

Fig. 1.

Overall survival of the entire cohort. Kaplan–Meier analysis representing overall survival following combined immunotherapy comprising intratumoral IL-2 and systemic ipilimumab treatment for 15 included patients. Censored patients are indicated by vertical lines

Correlative side studies

PBMCs were collected from all 15 patients at baseline. Additional samples were taken subsequently from 11 patients. PBMC samples for both follow-up time points were available after start of treatment for two patients, while only the early time point or only the late time point was available for 6 and 3 patients, respectively. Dynamic changes under treatment were analyzed for peripheral blood frequencies of immune cell populations, which were previously reported as biomarkers for patients treated with ipilimumab (Table 4). Treatment with ipilimumab in combination with intratumoral IL-2 resulted in an increase in absolute and relative numbers of lymphocytes in 13 and 12 of the 15 patients at the early time point compared to baseline (p = 0.003 and p = 0.036, respectively). Moreover, absolute eosinophil counts (AEC) and frequencies of Tregs were found to be significantly increased in all analyzed patients (p = 0.001 and p = 0.018, respectively). All these dynamic alterations were temporary and limited to the comparisons between the early time point during therapy and baseline in patients. No significant changes were observed comparing baseline and the late time point (Table 4).

Table 4.

Dynamic changes under treatment

Factor	Categories	Week 4 compared to baseline		Week 12 compared to baseline
		N (%)	p value	N (%)	p value
Absolute lymphocyte counts	Increase	13 (86.7)	0.003	5 (50.0)	0.721
	Decrease	2 (13.3)		5 (50.0)
Relative lymphocyte counts	Increase	12 (80.0)	0.036	4 (40.0)	1.00
	Decrease	3 (20.0)		6 (60.0)
Absolute eosinophil counts	Increase	15 (100)	0.001	7 (70.0)	0.114
	Decrease	0 (0.0)		3 (30.0)
Absolute monocyte counts	Increase	5 (33.3)	0.233	6 (60.0)	0.203
	Decrease	10 (66.7)		4 (40.0)
Lin−CD14+HLA-DR−/low MDSCs	Increase	3 (37.5)	0.263	3 (60.0)	0.225
	Decrease	5 (62.5)		2 (40.0)
CD4+CD25+FoxP3+ Tregs	Increase	7 (100)	0.018	1 (100)	n.a.
	Decrease	0 (0.0)		0 (0.0)

MDSCs myeloid-derived suppressor cells, Tregs regulatory T cells, n.a not applicable

At baseline, the following clinical characteristics and biomarkers were correlated with OS (Supplementary Table 2). Patients with AEC ≥ 50 counts/µL at baseline survived longer than patients with AEC < 50 counts/µL (p = 0.009; median survival 8.3 versus 2.5 months, respectively). No patient with low AEC survived the first year after start of treatment. Baseline frequencies of Lin^-CD14⁺HLA-DR^-/low MDSCs were negatively correlated with survival (p = 0.049). Median OS decreased from 30 to 9.9 to 3.3 months for patients with <5.1, ≥5.1 and ≥9.5% of MDSCs, while the risk of dying within the first 12 months increased from 33.3 to 50 to 85.7%, respectively. Recently, we described that the number of favorable baseline factors within the cluster comprising normal LDH, relative lymphocyte counts (RLC) ≥10.5%, AEC ≥ 50 counts/µL and absolute monocyte counts <650 counts/µL was strongly associated with survival [29]. Five patients (33.3%) with favorable values for all four parameters were identified in the present cohort. These patients had a high 1-year survival rate of 80% and a median survival of 30 months. In marked contrast, patients even with at least just one unfavorable factor had a short median survival of 4.1 months and 90% of these patients had died within the first year of treatment with IL-2 plus ipilimumab (Supplementary Table 2; p = 0.035). Analyzing correlations of changes of immune cell populations during treatment and OS showed that only an early increase in absolute lymphocyte counts and RLC upon treatment with ipilimumab and IL-2 were positively associated with OS (Supplementary Table 3). No other significant correlations between early or late changes in peripheral blood frequencies of immune cell populations and outcome were identified (Supplementary Tables 3, 4).

Discussion

Recently, the concept of intratumoral injection of drugs aiming in a beneficial systemic effect experienced a breakthrough by the Food and Drug Administration and European Medicines Agency approval of talimogene laherparepvec (T-VEC) for unresectable melanoma. T-VEC is a genetically modified herpes simplex virus type 1, which is injected intratumorally. In addition to its direct lytic activity, the release of GM-CSF in infected cells aims to initiate a systemically active anti-tumor immune response [30]. In a phase III trial patients treated with T-VEC had a significantly higher rate of durable responses (p < 0.001) and a strong trend toward longer median OS (p = 0.051) compared to those treated with subcutaneous GM-CSF in the control arm [31]. A phase IB trial testing the combination with systemic ipilimumab was promising [32] but results of a phase III trial comparing T-VEC in combination with ipilimumab versus ipilimumab monotherapy (NCT01740297) are not available yet.

In the current clinical trial, we investigated tolerability and efficacy of a combined immunotherapeutic strategy comprising the standard four intravenous doses of ipilimumab at 3 mg/kg every 3 weeks and intratumorally injected IL-2 applying 9 MIU daily twice per week for four weeks. The tolerability of this combined treatment was acceptable and within the range of expectations. Observed immune-related adverse events were similar to those described for ipilimumab monotherapy. Autoimmune colitis occurred in two patients (grade I in one and grade III in the other patient) but resolved under symptomatic and immunosuppressive treatment following recommendations for the management of immune-related adverse events [33]. Six patients developed a rash, which was asymptomatic in the majority of cases. Fatigue, flu-like symptoms, and fever were pronounced on the day of intratumoral IL-2 injections and were most likely attributable to these. Similar adverse events were previously reported for intratumoral IL-2 injections in stage III melanoma patients [4, 5]. No suspected unexpected serious adverse reactions and no treatment-related deaths occurred.

The disease control rate among the 15 patients was low at only 20% according to irRC, and only one patient (7%) had stable disease according to mWHO criteria. No objective responses were observed. In comparison, the disease control rate according to mWHO criteria was reported as 28.5% for pretreated patients in the ipilimumab monotherapy arm of the MDX-010 study and 20.1% for patients treated with ipilimumab and gp100 peptide vaccine in combination [10]. Similarly, no evidence to support a synergistic effect of CTLA-4 blockade plus systemic IL-2 administration was reported in an early phase I/II clinical trial [34]. Differences between the clinical characteristics do not serve as an explanation for the lower efficacy observed here, as the prognostic parameters were rather more favorable for the patients in our current study compared to the MDX-010 trial in terms of the Eastern Cooperative Oncology Group performance status, serum LDH and the M category according to AJCC [10]. Because efficacy criteria to support inclusion of additional patients in part two of the study (SD, PR, or CR in at least 6 of 15 patients) were not met in the protocol-defined interim analysis, the study was stopped after part 1. In line with the low proportion of patients experiencing clinical benefit, the 1-year-OS rate was 33.3% and lower than in the ipilimumab arms of the MDX-010 trial [10].

In correlative side studies, relative to baseline, we observed an increase in the frequency of CD4⁺CD25⁺FoxP3⁺Tregs in the peripheral blood 4 weeks after starting treatment in all 7 analyzed patients. An effect of IL-2 and/or ipilimumab on Tregs is not surprising as these cells express both the high affinity IL-2 receptor complex (consisting of CD25, CD122 and CD132) or CTLA-4 [14]. However, the clinical significance of this increase in the peripheral blood remains unclear. A prognostic impact of the frequency of Tregs in the peripheral blood was initially reported by Baumgartner and colleagues [35], who analyzed 14 patients with melanoma. However, we could not reproduce this finding in a study of 133 subjects [36]. In the peripheral blood, Treg increases during immunotherapy are not uncommon and were observed after intratumoral treatment with L19-IL-2, a cytokine-antibody construct with immunologic properties similar to the recombinant IL-2 used here [9], as well as after systemic high-dose IL-2 [37] and after ipilimumab monotherapy [21]. However, none of these studies reported associations between these increases of Tregs and poorer clinical outcome. Some studies even suggest a positive association of increased peripheral blood Treg frequencies and clinical outcome. Thus, Schwartzentruber and colleagues observed higher blood levels of Tregs in patients with clinical response after high-dose IL-2/vaccine treatment relative to those with progressive disease [38], and high frequencies at baseline were associated with positive outcome in patients treated with ipilimumab [29]. In tumor tissue, ipilimumab improves the ratio of intratumoral effector CD8⁺ T cells to FoxP3⁺ Tregs in responding cancer patients [39, 40], but no such association was observed in patients treated with the CTLA-4 antibody tremelimumab [41]. Further studies are needed to investigate whether the observed increases in circulating Tregs may be more pronounced on combined treatment compared to ipilimumab monotherapy and if this might result in any impaired clinical efficacy.

The combined immunotherapy described here resulted in dynamic increases in absolute eosinophil and lymphocyte counts and in RLC. All changes were temporary and seen only when analyzing samples taken 4 weeks after starting therapy, but were no longer detectable by 12 weeks, at the time of radiologic tumor assessment. The analysis of associations between baseline factors or changes in the abundance of immune cells was limited due to the low sample size and the low proportion of patients experiencing clinical benefit. Nevertheless, we observed a correlation between increases in RLC and OS. We had previously reported a trend toward a similar association in a study on ipilimumab monotherapy [21]. In the present study, an inverse correlation was observed between the frequency of MDSCs at baseline and OS, also as previously reported for advanced melanoma patients independently of type of treatment [36] as well as in patients under ipilimumab monotherapy [29]. Similarly, patients in the current study with high AEC had a better OS than those with an AEC < 50/µL, also in line with previous findings [29]. Despite the role of eosinophils in cancer immunity being largely unknown, similar findings were also reported by others for patients treated with ipilimumab [42] and for patients treated with pembrolizumab [43].

Based on our study of 15 patients, there was no evidence that parallel intratumoral treatment with IL-2 leads to improved efficacy compared to ipilimumab monotherapy. As efficacy was worse than expected considering historical data of patients treated with ipilimumab, and translational studies revealed changes in potentially relevant immune cell subsets (e.g., an increase in Tregs), one could speculate that the addition of intratumoral IL-2 might even have been detrimental. In fact, this cannot be excluded, but vice versa, due to the fact that only step 1 in the two-step design of the current trial was conducted, sample size is too small to support such a conclusion statistically. However, such a possibility has to be considered in future trials. This is of particular importance as similar combination approaches are currently being tested, e.g., the combination of intratumoral T-VEC and ipilimumab as described above or that of T-VEC with pembrolizumab (NCT02263508). In conclusion, immunotherapy combining intratumoral IL-2 injections with systemic ipilimumab is associated with manageable adverse events, which are similar to those associated with the respective monotherapies. However, clinical benefit was only observed in single patients. Thus, our results do not provide evidence for improved efficacy of the combined treatment approach compared to ipilimumab monotherapy.

Electronic supplementary material

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Abbreviations

AEC

Absolute eosinophil counts

AJCC

American Joint Committee on Cancer

CR

Complete response

irDCR

Immune-related disease control rate

irRC

Immune-related response criteria

MIU

Million international units

mWHO

Modified World Health Organization

PR

Partial responses

RLC

Relative lymphocyte counts

SD

Stable disease

Tregs

Regulatory T cells

T-VEC

Talimogene laherparepvec

Compliance with ethical standards

Conflict of interest

B Weide reports receiving commercial research grants from Bristol–Myers Squibb (BMS) and MSD Sharp and Dohme (MSD) and reports receiving travel/accommodations/expenses from BMS, MSD, Roche, Amgen, Philogen, Curevac and compensated advisory services for MSD, BMS, Philogen and Curevac. C. Garbe reports receiving honoraria from BMS, MSD, Amgen, Novartis, Roche, GlaxoSmithKline (GSK) and reports receiving commercial research grants from MSD, BMS, Roche, GSK. T.K Eigentler reports receiving honoraria from BMS, MSD, Roche and Novartis, travel/accommodations/expenses from BMS and is a consultant/advisory board member for BMS. No potential conflicts of interest were disclosed by the other authors.