Abstract
Phase I testing of the hu14.18-IL2 immunocytokine in melanoma patients showed immune activation, reversible toxicities, and a maximal tolerated dose of 7.5 mg/m2/day. In this phase II study, 14 patients with measurable metastatic melanoma were scheduled to receive hu14.18-IL2 at 6 mg/m2/day as 4-h intravenous infusions on Days 1, 2, and 3 of each 28 day cycle. Patients with stable disease (SD) or regression following cycle 2 could receive two additional treatment cycles. The primary objective was to evaluate antitumor activity and response duration. Secondary objectives evaluated adverse events and immunologic activation. All patients received two cycles of treatment. One patient had a partial response (PR) [1 PR of 14 patients = response rate of 7.1 %; confidence interval, 0.2–33.9 %], and 4 patients had SD and received cycles 3 and 4. The PR and SD responses lasted 3–4 months. All toxicities were reversible and those resulting in dose reduction included grade 3 hypotension (2 patients) and grade 2 renal insufficiency with oliguria (1 patient). Patients had a peripheral blood lymphocytosis on Day 8 and increased C-reactive protein. While one PR in 14 patients met protocol criteria to proceed to stage 2 and enter 16 additional patients, we suspended stage 2 due to limited availability of hu14.18-IL2 at that time and the brief duration of PR and SD. We conclude that subsequent testing of hu14.18-IL2 should involve melanoma patients with minimal residual disease based on compelling preclinical data and the confirmed immune activation with some antitumor activity in this study.
Keywords: Immunocytokine, Advanced Melanoma, Immunotherapy, Interleukin-2, Anti-ganglioside antibody, Phase II clinical trial
Introduction
Treatment of metastatic melanoma patients with high-dose bolus interleukin-2 (IL2) results in durable antitumor benefit in some patients [1]. Prior studies reported objective response rates of 16 %, complete response rates of 6 %, and noted that metastatic patients who responded for more than 30 months after high-dose IL-2 were unlikely to have subsequent disease recurrence [2]. Thus, improving IL-2 therapy has potential to significantly impact the management of metastatic melanoma patients. One approach to improve efficacy of IL2-based treatment is to focus the cytolytic activity of activated NK cells to mediate increased tumor-specific destruction [3, 4]. This can be demonstrated in vitro with the addition of monoclonal antibodies (mAb) to facilitate antibody-dependent cell-mediated cytotoxicity (ADCC). This approach has been attempted in clinical trials of anti-ganglioside mAb combined with IL2 [5]. GD2 is a disialoganglioside expressed on tumors of neuroectodermal origin including melanoma, neuroblastoma, and certain sarcomas. Expression on normal tissues is limited to the cerebellum, peripheral nerves, and very few other tissues. The relatively tumor selective expression of GD2 makes it a suitable target for mAb treatment. In vitro, anti-GD2 antibodies can mediate substantial ADCC and complement-dependent cytotoxicity (CDC) against GD2+ tumor target cells [4].
The original IgG3 murine anti-GD2 mAb 14.18 was developed by Dr. Ralph Reisfeld [6]. An IgG2a class switch variant of 14.18, called 14.G2a, was prepared in an attempt to mediate enhanced ADCC. This antibody has been tested as a single agent and in combination with IL2 in clinical trials. When antitumor activity was noted, a human/murine chimeric mAb ch14.18 was constructed using the murine variable genes of 14.18 and the human constant IgG1 and k genes, which are known to be effective at CDC and ADCC [7]. The ch14.18 antibody was tested in phase I clinical trials alone and in combination with IL2 in the treatment of melanoma patients. In an effort to improve the immunologically mediated antitumor activity, an immunocytokine (IC) composed of ch14.18 antibody and the cytokine IL2 was constructed by fusing a synthetic human IL2 gene sequence to the carboxyl end of the human Ig gene [8]. This ch14.18-IL2 IC has undergone extensive preclinical testing and has shown significant tumor reactivity and clinical potential [9–11]. Despite the previous observation of ADCC with anti-GD2 mAbs used in combination with soluble IL2, this mechanism is potentially limited by the number and type of available effector cells. Achieving ADCC depends upon the presence of effector cells with Fc receptors and upon the number of Fc receptors on the effector cells [12]. When NK cells with Fc receptors are activated and expanded with IL2 in vivo, they mediate dramatically augmented ADCC [4]. However, many of the activated NK cells circulating in cancer patients following in vivo treatment with IL2 do not have Fc receptors, in contrast to resting NK cells [13]. These Fc receptor negative, activated NK cells are more cytolytic in direct lytic assays not dependent on mAb and Fc receptors [13]. Thus, it would be advantageous to enable these activated NK cells to lyse tumor using a recognition mechanism that does not depend upon Fc receptors.
The in vivo activated NK cells have augmented expression of the IL2 receptor beta (IL2R-b) [14] and demonstrate a dramatic in vitro response to IL2 [15]. Furthermore, IL2R-bearing T cells that do not specifically recognize these tumors with their TCRs should still be responsive to IL2. Thus, it would be advantageous to activate these IL2 receptors with a molecule that will bridge the NK cells and T cells to tumor and activate lytic interactions. This is the proposed function of the ch14.18-IL2 IC. This molecule utilizes the 14.18 anti-GD2 antibody-mediated recognition component to bind to tumor cells, the Fc component to bind to cells expressing Fc receptors, and the IL2 component to activate lytic cells expressing IL2 receptors. Despite the substantial human component of the chimeric ch14.18 antibody, several patients treated with this antibody developed strong anti-idiotype antibody responses. The dose-limiting toxicities (DLT) also included allergic symptoms (angioedema) in some patients, potentially related to the murine component of this chimeric antibody [16, 17]. Hu14.18-IL2 was thus developed to maintain the immunologic activity of the ch14.18-IL2 IC molecule and decrease IC-related human anti-chimeric antibody (HACA) responses and allergic reactions. S. Gillies and colleagues utilized the same technology used to create chl4.18-IL2 [8] for the construction of hu14.18-IL2. The hu14.18 Ab portion contains only the complementarity-determining regions (CDR) of the murine variable chains, grafted into the intact human IgG1 molecule, which has an IL2 molecule at each carboxy terminus of the IgG1 heavy chains. Our phase I clinical trial of hul4.18-IL2 in adults with melanoma showed that hu14.18-IL2 is clinically safe and well tolerated at doses (0.8–7.5 mg/m2) that induce immunologic activation [18]. A total of 33 patients with melanoma were enrolled to establish the maximal tolerated dose (MTD). Patients were administered hu14.18-IL2 at one of the following dose levels: 0.8, 1.6, 3.2, 4.8, 6.0, or 7.5 mg/m2/day. Hu14.18-IL2 was administered as a 4-h IV infusion over 3 consecutive days during the first week of each course. The dose of 7.5 mg/m2/day was found to be the MTD, as two of six subjects showed reversible dose-limiting toxicity at this dose level. These included hypoxia, hypotension, and elevations of AST and ALT [18].
The primary objective of this phase II study of hu14.18-IL2 in advanced melanoma patients was to evaluate the clinical antitumor activity and duration of response at one dose level beneath the previously determined MTD. Secondary objectives included evaluating adverse events and immunologic activation. The hu14.18-IL2 dose for this phase II trial was 6 mg/m2 and was one dose below the MTD of 7.5 mg/m2 established in our phase I trial [18]. The dose of 6.0 rather than the 7.5 mg/m2/day was chosen, as the MTD determination in the phase I study was based on DLT events in the first course of treatment. However, most patients in this study were expected to be receiving four courses of treatment. As some patients in the adult and pediatric phase I studies did have episodes of DLT in subsequent courses of treatment not necessarily seen in the first course, it was determined that starting at the 6.0 mg/m2/day dose would be more likely to enable patients to continue on treatment without requiring dose reductions.
Materials and methods
Clinical protocol
Patients
Fourteen patients were enrolled and all patients provided written informed consent. Enrollment required patients to have histologically confirmed malignant melanoma that was considered surgically and medically incurable and was measurable by clinical assessments or imaging. All patients needed to have adequate hematologic parameters (Total WBC ≥3,500/mm3, platelets ≥100,000/mm3, and hemoglobin ≥10.0 g/dl), adequate liver function (AST/ALT <2 times normal and a total bilirubin <2.0 mg/dl), and adequate renal function (serum creatinine <2.0 mg/dl or a creatinine clearance of ≥60 ml/min). All patients had an Eastern Cooperative Oncology Group performance status of 0 or 1. Patients with a history of cardiac disease, age ≥65 years old, or with significant risk factors for coronary artery disease were required to complete an exercise radionuclide scan with no evidence of myocardial ischemia or heart failure and have normal pulmonary function. Criteria for patient exclusion included exclusion of patients who had previously received corticosteroids or other immunosuppressive therapy within 2 weeks before the study entry. Patients with CNS metastases were also excluded. Patients who previously received mAbs for any reason and had detectible antibody (over background) to hu14.18 were excluded. Patients with a history of diabetes mellitus that required systemic therapy within the past 3 months were excluded, as this therapy may alter blood glucose levels. Patients with HIV or hepatitis B surface antigen carrier state or with clinical evidence of hepatitis were ineligible. Patients who had received any (standard or experimental) systemic therapy for stage IV disease were excluded.
Hu14.18-IL2
The hu14.18-IL2 was supplied collaboratively by the NCI-Biological Resources Branch (NCI-BRB, Frederick, MD) as well as EMD Pharmaceuticals (Durham, NC) and Merck Serono (Darmstadt, Germany). Preclinical evaluation has shown that 1 mg of the fusion protein contains approximately 3 × 106 U of IL-2 (based on a proliferative assay with IL-2 responsive Tf-1 beta cells) and approximately 0.8 mg of the hu14.18 mAb [19].
Study design
This was a two-stage study design. Initially, 14 patients were to be enrolled and receive hu14.18-IL2 on Days 1, 2, and 3 of each course of therapy as a 4-h continuous intravenous infusion at a daily dose of 6 mg/m2/day. Patients could receive a maximum of four 28-day courses of treatment at the same dose level, provided that there was no dose-limiting toxicity (DLT) or significant non-DLT toxicity. The protocol allowed for two dose modifications for DLT or significant non-DLT toxicity: first, a 50 % dose reduction to 3 mg/m2/day, and the second, an additional 50 % dose reduction to 1.5 mg/m2/day. Missed doses were not made up, and all dose reductions were permanent. If there were no responses in these 14 subjects (either complete or partial), the study would be closed due to the lack of clinical efficacy. If a response was seen (complete or partial) in at least one subject, an additional 16 subjects could be enrolled depending on the availability of the hu14.18-IL2. If insufficient clinical material was available at that time, after discussion with the NCI-BRB (the producer of the hu14.18-IL2) and EMD (the licensor for hu14.18-IL2 and MCRADA holder with the NCI, at the time of this study), then the study would be closed to accrual. If sufficient material was available, however, 16 additional patients could be enrolled at the same dose level. If four or more of the 30 subjects had objective responses, the protocol regimen would be considered to have sufficient clinical activity to warrant further study. All eligible subjects who receive any administration of hu14.18-IL2 are considered evaluable for efficacy and safety.
Toxicity grading and dose modifications
The definition of DLT was defined as grade 3 or 4 toxicity, using the NCI Common Terminology Criteria Adverse Events (CTCAE), version 3.0, except for the following which are known side effects of IL-2 and hu14.18-IL2 therapy: grade 3 pain, grade 3 nausea and vomiting, grade 3 fever (Temp >40 °C), grade 3 skin toxicity, grade 3 metabolic/laboratory toxicity of hyponatremia, hyperglycemia, or hypophosphatemia in the absence of CNS symptoms, grade 3 sensory or motor peripheral (non-cranial) neuropathy if transient and reversing within 3 days of completion of hu14.18-IL2, grade 3 hematologic toxicity (or grade 4 lymphopenia-a known transient marker of immune activation), grade 3 infusional reactions lasting less than 24 h, grade 3 fatigue or ECOG performance status change that resolves in ≤1 week, and grade 3 infection that resolves in ≤1 week. These toxicities, if seen, were graded and recorded but not used as criteria for determining DLTs as they were expected, based on the observations of hu14.18-IL2 given to 33 adults with melanoma, administered by the same schedule at similar doses to that being tested in this trial [18].
All grade 4 toxicities, with the exception of transient lymphopenia, were considered DLT and required stopping of treatment. If this toxicity was related to the hu14.18-IL2, the patient was removed from the study without further treatment. If the grade 4 toxicity was not related to the hu14.18-IL2 and resolved to baseline in less than 3 days, treatment was continued at 50 % of the daily dose from the previous course. All other DLT and significant non-DLT toxicity had therapy held and resumed at 50 % of the initial dose once the toxicity resolved. Missed doses were not given at a later date. If toxicity again required treatment cessation, the dose was reduced to 25 % of the initial dose. If treatment at this reduced dose resulted in recurrence of the DLT or significant non-DLT toxicity, treatment was permanently discontinued.
Response criteria
Serial clinical measurements of tumor size were performed by standard radiologic and physical exam measurements, appropriate for each patient’s clinical status. Tumor measurements were scheduled to take place after every two courses of treatment and at the completion of protocol treatment. The clinical response to treatment was determined at each of the disease status evaluation time points. The NCI Response Evaluation Criteria in Solid Tumors (RECIST) was used. A complete response was defined as a disappearance of all target lesions. A partial response was defined as at least a 30 % decrease in the sum of the longest diameters of target lesions. Progressive disease was defined as at least a 20 % increase in the sum of the longest diameters of the target lesions, taking as reference the longest diameters recorded at baseline. Stable disease was defined as neither sufficient shrinkage to qualify for partial response nor sufficient increase to qualify for progressive disease.
Immunologic monitoring
Serum samples were obtained from all patients to measure hu14.18-IL-2 levels, anti-hu14.18-IL2 antibodies, and soluble IL2 receptor α levels. Serum samples were drawn at baseline and on Days 1, 3, 4, 8, and 29. Blood samples were also collected from selected patients, at times shown, to evaluate C-reactive protein (CRP) levels and functional NK and ADCC assays. All assays shown (except CRP) involve ELISA methods or in vitro cell-mediated cytotoxicity assays as previously published [16, 18, 20–22]. The anti-idiotype bridge assay was considered positive for samples with OD >0.6; borderline for samples with OD from 0.3 to 0.6, and negative for samples with OD <0.3 [22]. The anti-idiotype binding inhibition assay was considered positive for samples showing >28 % inhibition [22]. The CRP assay was performed by the UW Hospitals and Clinics clinical lab, and the other laboratory assays used to evaluate DLTs per the CTCAE 3.0 criteria were standard clinical laboratory assays.
Statistical analysis
Treatment effects were assessed as the change in parameter varies from baseline to various times during treatment. The clinical response to treatment was determined at each of the disease status evaluation points. Serum IC level, changes in absolute lymphocyte count (ALC) from baseline to C1D3 and C1D8, and changes in CRP from baseline to C1D3 and C1D4 between those with best response of PD and PR/SD and sIL2R levels and absolute CRP levels at C1D4 and C2D4 between patients with and without DLTs were compared using two-sample Wilcoxon rank sum test. Changes in peripheral blood lymphocyte counts from baseline to Days 3 and 8 for both cycles 1 and 2 were evaluated using a paired t test. All p values are two-sided, and levels of p < 0.05 were considered as statistically significant. Data analysis was conducted suing SAS software version 9.2 (SAS Institute, Cary, NC).
Results
Patient characteristics
Fourteen patients were entered into this study and their pretreatment characteristics are outlined in Table 1. Eight of the 14 patients were men, the median age was 60 years, and all patients were ECOG performance status 0 or 1. Five patients had received previous adjuvant immunotherapy and 1 had received previous adjuvant biochemotherapy. Thirteen of the patients had a primary melanoma site on the skin, and one patient did not have a documented primary site. The most common sites of metastatic disease were lung (11 patients), liver (9 patients) and lymph node or other soft tissues (9 patients).
Table 1.
Patient characteristics (N = 14)
Characteristics | Number of patientsa |
---|---|
Sex | |
Male | 8 |
Female | 6 |
Site of primary disease | |
Skin | 13 |
Unknown | 1 |
Prior therapy | |
Surgery | 13 |
Immunotherapy | 5 |
Biochemotherapy | 1 |
Radiation therapy | 3 |
Sites of disease | |
Lymph node, subcutaneous, and other soft tissues | 9 |
Lung | 11 |
Liver | 9 |
Other visceral metastases (spleen, pancreas, adrenal) | 5 |
Bone | 1 |
AJCC disease classificationb | |
M1a | 0 |
M1b | 2 |
M1c | 12 |
Performance statusc | |
0 | 13 |
1 | 1 |
aMedian age, 60; range, 21–78
bAmerican Joint Committee on Cancer
cEastern Cooperative Oncology Group Performance Status
Treatment summary and antitumor effects
The treatment summary of patients in this study is shown in Table 2. All 14 patients received at least two cycles of hu14.18-IL2. Five patients were eligible for four cycles of therapy because of stable disease (four patients) or partial response (one patient). The other 9 patients had objective disease progression at the initial assessment of clinical response following cycle 2. The duration of stable disease or partial response was measured in months starting from the date of the clinical response assessment following cycle 2. The duration of stable disease ranged from 3 to 4 months, and the duration of the one partial response was 3.5 months. Three patients required dose reductions, and a modified dose of hu14.18-IL2 was administered in six of the 38 treatment cycles.
Table 2.
Treatment summary
Patient # n = 14 | Number of treatment cycles | Dose reductions (# required) | Clinical response after cycle 2 | Clinical response after cycle 4 | Duration of best response (months)a |
---|---|---|---|---|---|
1 | 2 | 0 | Progression | ||
2 | 2 | 0 | Progression | ||
3 | 2 | 0 | Progression | ||
4 | 2 | 0 | Progression | ||
5 | 4 | 1 | Stable disease | Stable disease | 4 |
6 | 4 | 0 | Stable disease | Stable disease | 3 |
7 | 4 | 1 | Stable disease | Stable disease | 4 |
8 | 2 | 0 | Progression | ||
9 | 2 | 0 | Progression | ||
10 | 4 | 0 | Stable disease | Stable disease | 4 |
11 | 2 | 0 | Progression | ||
12 | 2 | 0 | Progression | ||
13 | 4 | 1 | Partial response | Partial response | 3.5 |
14 | 2 | 0 | Progression | ||
Totals | 38b | 3 |
Partial response, n = 1 stable disease, n = 4 progression, n = 9 |
Partial response, n = 1 stable disease, n = 4 |
aDuration of best response was measured in months from the time the response was detected after course 2 to the time of objective disease progression
bModified dose was given in 6 of the 38 treatment cycles
Toxicities
Toxicities requiring dose reductions are shown in Table 3a and include hypotension refractory to brief intravenous fluids (2 patients) and grade 2 renal insufficiency with oliguria (1 patient). All three of these patients were able to receive the remainder of planned hu14.18-IL2 infusions with a 50 % dose reduction. The anticipated IL-2 constitutional symptoms (fever, chills, fatigue, pruritis, and myalgias) were seen in the majority of patients treated on this clinical trial. Two patients had severe fatigue and decline in performance status, but returned to baseline after the conclusion of the therapy. Several patients experienced pain associated with infusion of hu14.18-IL2 and required opioids for adequate pain control. All treatment-associated toxicities experienced by patients in this study were reversible.
Table 3.
Toxicities and laboratory changes observed during treatment
a. Toxicities requiring dose reduction | |
---|---|
Patient # (n = 3) | Toxicity |
5a | Hypotension refractory to brief IV fluids |
7b | grade 2 renal insufficiency with oliguria |
13c | Hypotension refractory to brief IV fluids |
3b. Treatment-related severe laboratory changesd,e | ||
---|---|---|
Toxicity | Grade 3 toxicityf | Grade 4 toxicityf |
N | N | |
Lymphopenia | 4 | 9 |
Thrombocytopenia | 3 | 0 |
PT/PTT | 1g | 0 |
Hypophosphatemia | 5 | 0 |
Hyponatremia | 1 | 0 |
Hypokalemia | 1 | 0 |
Hypocalcemia | 1 | 0 |
Hyperglycemia | 2 | 0 |
Hypermagnesemia | 1 | 0 |
aCycle 4 Day 3 therapy partially administered at 50 % dose reduction. Infusion stopped after 15 min due to recurrent hypotension
bCycle 3 Day 3 therapy held. Cycle 4 therapy administered at 50 % dose reduction
cCycle 2 Day 2 therapy held. Cycle 2 Day 3 and cycles 3 and 4 therapy administered at 50 % dose reduction
dAll toxicities were reversible
eThe majority of patients had transient grade 1 and 2 laboratory changes including neutropenia, anemia, thrombocytopenia, hyperbilirubinemia, elevated AST, hypophosphatemia, and hyperglycemia
fTable provides the highest grade for each indicated toxicity
gUnrelated to protocol therapy
The grade 3 and 4 laboratory toxicities experienced by patients in this study are outlined in Table 3 b. The only grade 4 laboratory toxicity was a transient grade 4 lymphopenia associated with hu14.18-IL2 infusions and was similar to what we previously reported with hu14.18-IL2 infusions [18]. All grade 3 laboratory changes were reversible and did not require a dose reduction. Many patients had transient grade 1 and 2 laboratory changes that were similar to those reported in our prior phase I study of hu14.18-IL2 [18].
Immunologic results/correlative studies
Hu14.18-IL2 levels
Analysis of serum IC for all patients showed a mean value of 1.42 μg/ml on C1D1 and 1.38 μg/ml on C1D3. The slightly decreased level of IC on Day 3 (compared to Day 1) is expected and was also seen in a separate Children’s Oncology Group study of patients with neuroblastoma [20]. No correlation was found between peak IC level on Day 1 and any toxicity parameter or clinical response. The peak level of IC on Day 1 did not differ significantly between patients who had progressive disease and who that had stable disease or a partial response (Table 4a). There was also no association between peak IC level and the following parameters: absolute drop in lymphocyte count from baseline to the nadir (Day 3), absolute rise in lymphocyte count from baseline to the lymphocyte rebound seen on Day 8, increase in soluble IL2 receptor levels, or change in CRP (not shown).
Table 4.
Laboratory correlative studies
a. Serum IC level by best response | |||||
---|---|---|---|---|---|
Best response PD | Best response SD or PR | ||||
N | Mean mcg/ml (SD) | N | Mean mcg/ml (SD) | p valuea | |
C1D1 | 9 | 1,303.3 (447.6) | 5 | 1,631.6 (460.5) | 0.217 |
C1D3 | 9 | 1,130.7 (1,003.6) | 5 | 1,371.8 (550.6) | 0.632 |
C2D1 | 9 | 1,399.1 (405.3) | 5 | 1,350.4 (480.9) | 0.842 |
C2D3 | 9 | 1,279.9 (820.0) | 5 | 1,399.8 (650.3) | 0.786 |
b. Change in absolute lymphocyte count (cells/mcl) from baseline to C1D3 or C1D4 by best response | |||||||
---|---|---|---|---|---|---|---|
Best response PD | Best response SD, PR, or CR | ||||||
N | Mean (SD) | p valueb | N | Mean (SD) | p valueb | p valuec | |
Baseline to C1D3 | 9 | −1,162 (598) | <0.001 | 4d | −1,561 (455) | 0.006 | 0.226 |
Baseline to C1D8 | 9 | 1,851 (964) | <0.001 | 4d | 2,362 (1,548) | 0.055 | 0.476 |
c. Comparison of sIL2R levels between patients with DLTs versus no DLTs | |||||
---|---|---|---|---|---|
DLT | No DLT | ||||
N | Mean pg/ml (SD) | N | Mean pg/ml(SD) | p valuee | |
C1D4 | 3 | 6,173 (2,286) | 11 | 6,595 (2,123) | 1.0 |
C2D4 | 3 | 8,098 (2,803) | 11 | 7,186 (1,884) | 0.6 |
d. Change in CRP (mg/dL) from baseline to C1D3 or C1D4 by best response | |||||||
---|---|---|---|---|---|---|---|
Best response PD | Best response SD or PR | ||||||
N | Mean (SD) | p valuef | N | Mean (SD) | p valuef | p valueg | |
Baseline to C1D3 | 9 | 11.2 (4.4) | <0.001 | 4 | 11.3 (5.7) | 0.03 | 0.993 |
Baseline to C1D4 | 3 | 10.3 (3.1) | 0.028 | 3 | 14.3 (6.0) | 0.054 | 0.363 |
e. Comparison of absolute CRP levels (mg/dl) between patients with DLTs vs no DLTs | |||||
---|---|---|---|---|---|
DLT | No DLT | ||||
N | Mean (SD) | N | Mean (SD) | p valueh | |
C1D4 | 2 | 19.5 (6.36) | 5 | 10.6 (2.41) | 0.081 |
C2D4 | 2 | 18.0 (1.41) | 5 | 11.0 (2.45) | 0.079 |
aNon-parametric Wilcoxon rank sum test (two-tailed) between response groups
bPaired t test for evaluating changes from baseline within groups
cNon-parametric Wilcoxon rank sum test (two-tailed) between response groups
d4 patients, rather than 5 patients, in this group as one patient had sample obtained at incorrect time
eNon-parametric Wilcoxon rank sum test (two-tailed) between response groups
fPaired t test for evaluating changes from baseline within groups
gNon-parametric Wilcoxon rank sum test (two-tailed) between response groups
hNon-parametric Wilcoxon rank sum test (two-tailed)
Immune activation
Figure 1 shows immune activation as measured in a 51Cr release assay. Killing of M21 melanoma cells was examined in the presence of serum taken from each of the 14 patients just before or just after the 4 h IC infusion on Day-1 of course 1. This assay was done in the presence or absence of PBMCs from a healthy donor. The serum from the patient was not heat treated, to enable endogenous complement to mediate killing if an appropriate antibody was present. The first column in Fig. 1 shows that virtually no killing is mediated against M21 melanoma cells by preinfusion serum from these 14 patients. Addition of PBMCs from a healthy donor (column 2 in Fig. 1) does not induce detectible cytotoxicity. In contrast, when serum obtained just after the IC infusion was tested in this assay, in the absence of PBMCs (column 3 in Fig. 1), six of the 14 patients showed detectible tumor cell killing. This indicates that sufficient antitumor antibody (the IC infused over the prior 4 h) and complement was present in the serum from those 6 patients to enable complement-mediated tumor cell lysis that was not observed with the pretreatment serum sample. More importantly, for all 14 patients, when healthy PBMCs were added as effector cells to the serum obtained after the 4 h infusion, an increase in killing was seen over that mediated by those same effector cells with the patients’ pretreatment serum (comparing columns 2 and 4 in Fig. 1), documenting the acquisition of conditions able to induce ADCC. The fact that these same effector cells do not induce any detectible killing when the source of serum is obtained from these same 14 patients immediately before the IC infusion (again, compare columns 2 and 4 in Fig. 1) indicates that the infusion of the IC into these patients has enabled their serum to mediate ADCC, using the circulating IC (detected by ELISA and shown in Table 4a). Furthermore, the addition of these PBMCs to the 4-h serum sample caused an increase in killing over that observed by this same serum without effectors, for all 14 patients (compare columns 3 and 4). This demonstrates that the killing observed in the presence of PBMCs is greater than that which can be mediated via complement with this same serum sample and thus reflects ADCC.
Fig. 1.
Antibody-dependent cell-mediated cytotoxicity (ADCC) with hu14.18-IL2 in patient serum. The M21 human melanoma target (% cytotoxicity; effector / target ratio of 50:1) was evaluated with (+) and without (−) effector PBMC from a healthy control donor in medium containing patient serum obtained pretreatment (pre) or after the initial 4 h hu14.18-IL2 infusion (4 h)
Figure 2 shows the lymphopenia that was anticipated on Day 3 of each course that was followed by a lymphocytosis, as previously described [18, 20]. The magnitude of the drop in ALC on Day 3 was not different for the 9 patients with PD than for the 4 evaluated patients with SD/PR (Table 4b). Neither was the rebound increase in ALC seen on Day 8 (Table 4b).
Fig. 2.
Mean peripheral blood lymphocyte count (cell number per mcl ± SD) at the indicated protocol day for the 14 patients in this study. Time points are pretreatment (Pre) and at the indicated cycle (C) and day (D) of that cycle. Changes from Pre were significant for C1D3, C1D8, C2D3, C2D26 (p < 0.001), and C1D26 (p = 0.011)
As previously seen, we observed a significant increase in soluble IL2 receptor (sIL2R) levels in all courses from baseline to Day 3 (p < 0.001 for both courses 1 and 2) [18, 20]. Table 4 c shows the increase in sIL2R levels was not different for those 3 patients with DLT versus those 11 without. Similarly, there was no difference in sIL2R level for the 5 with PR or SD versus the 9 patients with progressive disease (not shown). CRP levels are a common clinical laboratory parameter for immune activation. As expected [20], we found this parameter was increased by treatment with hu14.18-IL2 (as detected on the 3rd and 4th day of each course). As this laboratory assay was not required initially for all patients, we do not have a complete data set of values for all patients at all time points. Even so, the increased values over baseline are seen in Table 4d, e. Table 4d shows that there is no difference in the CRP response for the patients with PD versus those with SD/PR. It is of interest that the trend was for CRP levels on Day 4 of both course 1 and course 2 to be higher for the patients with DLT than those without (Table 4e; p = 0.08), consistent with the DLT being due to the immune activation (likely from the IL2 component of the IC).
Anti-hu14.18-IL2 antibody
In previous studies [20, 21], we have shown that some patients develop antibodies to the idiotypic determining region of the IC; these antibodies are measured by a bridging assay or a binding inhibition assay [22]. In this phase II study of 14 evaluable patients, 8 patients developed a “positive” anti-idiotype antibody against hu14.18-IL2 based on the bridging assay and 13 developed a detectible anti-idiotypic antibody based on the “binding inhibition” assay. The development of these anti-IC antibodies was not associated (statistically) with the peak level of IC, for these 13 patients in this study (not shown). Furthermore, there was no significant effect of this anti-idiotypic antibody response on the in vivo level of hu14.18-IL2. Specifically, there was no significant association of the level of anti-idiotypic antibody developed after course-1 (p = 0.06 for the bridge assay and 0.6 for the binding inhibition assay) with any detectible decrease in peak hu14.18-IL2 level seen on day-1 of course-2 (compared to the level seen on day-1 of course-1). This lack of a significant association is in contrast to the significant drop in hu14.18-IL2 levels from course-1 day-1 to course-2 day-1 for those patients with positive anti-idiotype responses (in the “binding inhibition” (p = 0.002) assay) in our past phase I trials (where most patients received lower IC doses) [22].
Discussion
New insights are critically needed to achieve durable benefit for patients with metastatic melanoma, as this cancer is usually incurable once metastatic to distant sites. Recent advances provide new options for metastatic melanoma patients. Ipilimumab and vemurafenib were approved in 2011 based on their proven benefit of improving the survival of some patients with metastatic melanoma [23–25]. While vemurafenib can induce complete or partial tumor regression in up to 80 % of metastatic melanoma patients with the V600E BRAF mutation, those responses are of limited duration for the overwhelming majority of treated patients [25]. Response rates with Ipilimumab are only in the 10–20 % range, but those response can be durable for years [23, 24]. Therapy with high-dose bolus interleukin-2 also provides durable benefit for a minority of metastatic melanoma patients [7]. The clinical challenge remains identifying treatments that can achieve durable benefit for a greater number of metastatic melanoma patients.
This protocol examined the treatment of advanced melanoma patients with hu14.18-IL2 at one dose level beneath the previously determined MTD as 4-h IV infusions over 3 consecutive days during the first week of each 28-day course of therapy. Treatment with hu14.18-IL2 at this dose and schedule (6 mg/m2/day for three consecutive days) is well tolerated and has toxicities that are reversible and readily managed. Of the 14 patients who were treated, only 3 patients required dose reductions due to toxicities. It is of interest that the three patients who required dose reductions due to treatment-associated toxicities had either stable disease (2 patients) or a partial response (1 patient) that lasted up to 4 months. We considered the hypothesis that these patient toxicities were due to a more pronounced activation of their immune system. However, comparing laboratory parameters of immune activation including sIL2R, CRP, and absolute change in lymphocyte count between patients with PD versus SD/PR did not reveal significant differences. Immune activation was found in all treated patients as evidenced by rebound lymphocytosis, elevated CRP, increased sIL2R levels, and ADCC. Thus, general measures of immune activation did not predict PR or SD for patients in this study.
The objective response rate for unselected advanced melanoma patients receiving hu14.18-IL2 at 6 mg/m2/day, one dose level beneath the MTD, was 7.1 % (confidence interval, 0.2–33.9 %). Only one patient of the 14 enrolled had an objective partial response. While the confidence interval for this response rate is wide, none of the patients in this trial had durable benefit, as the longest duration for either stable disease or patient response was only 4 months. Due to this fact combined with the limited supply of the hu14.18-IL2 at that time, we concluded that there was not sufficient evidence to pursue the treatment of unselected advanced melanoma patients with this dose and schedule of hu14.18-IL2, and we canceled plans for stage 2 of this study.
In our phase I trials of hu14.18-IL2 [18, 20], we found that development of a detectible antibody response against the hu14.18-IL2 IC was associated with a significant decrease in the peak IC level seen in subsequent courses [22]. Such a relationship was seen in 11 melanoma patients receiving 2 courses of IC in the phase I trial [18]. In contrast, in this present study of 14 melanoma patients, there was no statistically significant influence of anti-IC antibody on the levels of IC detected in serum in subsequent courses. We hypothesize that even though we can detect anti-IC antibody in some IC-treated patients, the “neutralizing” effect of such antibody is not sufficient to influence the detected serum levels of IC for patients receiving the higher dose of IC received in this phase II trial (compared to the lower doses of IC used for most patients in the phase I melanoma trial) [18]. Recent in vitro data from our laboratory simulating the in vivo concentrations of IC and anti-IC antibody in the sera of these patients are consistent with this conclusion [Hank et al. manuscript in preparation].
In contrast to the observed limited activity of hu14.18-IL2 for patients with advanced melanoma seen in this phase II study, there have been encouraging data for this agent in the treatment of children with advanced (relapsed or refractory) neuroblastoma. Clear antitumor activity has been observed in our phase II trial [21]. However, in that phase II trial, antitumor activity was not observed in children with bulky disease (measurable by MRI or CT scans), but was only seen in children with “less bulky” disease; neuroblastoma evaluable only by bone marrow histology or 123I-MIBG scintigraphy [21]. These clinical data, showing greater antitumor activity in the setting of less bulky disease, were consistent with our preclinical data demonstrating greater activity of hu14.18-IL2 in mice with smaller amounts of tumor at the time of treatment [26]. As our preclinical data [26] and our clinical data from our study of children with neuroblastoma both suggest that hu14.18-IL2 may demonstrate greater efficacy in the minimal residual disease state, we are now studying the administration of hu14.18-IL2 in adult patients with recurrent stage III or IV melanoma who can be rendered free of all known diseases with surgical resection.
As our preclinical data indicated that a mechanism for IC-induced antitumor efficacy was via NK cells mediating ADCC [27, 28], we also evaluated the influence of receptors on cells of the innate immune system for their potential involvement in the antitumor activity seen in our phase II neuroblastoma trial [21]. Our clinical data demonstrated that individuals with favorable killer immunoglobulin-like receptor (KIR) and KIR-ligand genotypes were more likely to benefit from IC treatment [28]. In addition, there was a trend toward benefit for individuals with favorable Fc receptor genotype (for CD32) [28]. Further evaluation of KIR/KIR-ligand and FcR genotypes may be helpful in future studies of this hu14.18-IL2 in melanoma and of other immunotherapies that act, in part, via ADCC.
The current study did not require histological demonstration of GD2 expression for study eligibility. It is possible that GD2 expression by melanoma patients in this study was less than anticipated. We are testing GD2 expression of resected melanoma in the current study of hu14.18-IL2 for patients with recurrent stage III or stage IV melanoma who can be rendered free of all known disease with surgical resection. In addition, the demonstrated immunologic activation of hu14.18-IL2 with manageable toxicities suggests the potential for combination treatments of advanced melanoma patients with hu14.18-IL2 and other agents. Concepts that merit consideration include the combination of hu14.18-IL2 with experimental anti-melanoma vaccines, combination of hu14.18-IL2 with other immunotherapies such as Ipilimumab, and combination of hu14.18-IL2 with targeted therapies such vemurafenib. In addition, our preclinical data demonstrate more potent local and systemic effects can be obtained when hu14.18-IL2 is given as an intratumoral injection [29]. As other immunotherapies are being applied via intratumoral administration for testing in melanoma [30], we are working toward a future clinical trial of hu14.18-IL2 as intratumoral therapy in patients with melanoma.
Acknowledgments
We thank Barrett P. Wagner for technical assistance and Laddie Johnson for assistance with manuscript preparation. We also thank the nurses on the UW CTRC for outstanding nursing care and for clinical trial support. This material was supported by National Institutes of Health Grants CA032685, CA87025, and grants from the Midwest Athletes for Childhood Cancer Fund, the Crawdaddy Foundation; CTRC grant (GM067386); grant P30 CA014520 from the National Cancer Institute; the Gretchen and Andrew Dawes Melanoma Research Fund; Ann’s Hope Foundation; the Jay Van Sloan Memorial from the Steve Leuthold Family; and the Tim Eagle Memorial.
Conflict of interest
The authors have the following financial or other conflicts of interests to disclose related to this publication: The commercial rights to hu14.18-IL2 IC are now held by Apeiron-Biologics AG of Vienna Austria. They were licensed from Merck Serono of Darmstadt Germany. A separate (subsequent and ongoing) trial of hu14.18-IL2 in patients with melanoma, chaired by Dr. Mark Albertini and involving this clinical team, received partial per patient research support for components of the clinical study from Merck Serono. No direct support from Merck Serono was received regarding the study reported on here. Dr. Gillies has a patent related to hu14.18-IL2 that he has licensed to Merck Serono.
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