A total of 28 patients were enrolled, of whom 27 were fully evaluable for toxicity, and all 28 were evaluable for response. Pretreatment characteristics are outlined in . Twenty-seven of the enrolled patients had neuroblastoma, and one patient had melanoma. There were 13 (46.4%) male and 15 (53.5%) female patients. The median age of the 28 patients at the time of study entry was 6.63 years (range, 2.5–17.7 years), and all had performance status scores by Lansky or Karnofsky of ≥90. Prior therapies included surgery (23 patients), biological therapy (4 patients), radiotherapy (24 patients), and chemotherapy (28 patients). Eighteen patients had prior autologous bone marrow transplant. All patients had metastatic, stage IV disease at the time of study entry, and the mean time between diagnosis and study entry was 1.7 years. The dose escalation schedule is shown in . There were four patients treated at dose level 4 (8 mg/m2/d) because one patient was given dexamethasone for grade 2 allergic symptoms, making this patient ineligible for further toxicity assessment. This patient, during the first day of infusion, developed hives and was treated with 10 mg/m2 dexamethasone as opposed to benadryl as directed by the protocol. This required an additional patient be enrolled at that dose level to adequately evaluate toxicity.
Clinical toxicities grade ≥2 are detailed in . Twenty-six (93%) patients had grade 2 or 3 fevers. Eighteen (64%) patients had grade 2 or 3 pain (rectal, pelvic, myalgia, neuropathic, abdominal, arthalgia, chest, bone, and headache). Seventeen patients (61%) experienced grade ≥2 hypotension. Of the 17 patients with hypotension, three were grade 3 and one was grade 4 (). The latter patient (8 mg/m2/d) had two separate episodes of hypotension in courses 2 (grade 3) and 4 (grade 4). Two patients required dopamine to support systemic blood pressure, and the remaining two responded to i.v. fluids.
Clinical toxicities observed with hu14.18-IL2
Characteristics of patients with grade 3 or 4 hypotension
Toxicities determined by clinical lab evaluation as grade ≥2 are listed in . The majority of patients (68%) had grade 2 or 3 anemia. Eight patients (29%) had grade 2 or 3 neutropenia, and three patients had grade 4. Ten patients (36%) had thrombocytopenia. The majority of these were grade 2 or 3 with one grade 4 toxicity. Six patients (21%) experienced grade 3 and 4 lymphopenia, a known immune effect of IL-2.
Laboratory changes observed with hu14.18-IL2
Although the MTD was determined by DLT in course one only, DLT was evaluated in each course of therapy for each patient. If a patient experienced a DLT in any course, if eligible for further therapy, there was a dose reduction for future courses. During course 1, one patient required 50% dose reduction on day 2 of infusion and omission of day 3 due to an anaphylactic reaction. This patient was assigned to the 12 mg/m2/d dose level at study entry and only received one course of therapy. Six patients (30% of patients completing two courses of therapy) required dose modifications during course 2. One patient at 2 mg/m2/d received 50% dose reduction for course 2 and the subsequent two courses due to neutropenia. One patient assigned to 8 mg/m2/d (patient 13 in ) required 50% dose reduction on day 3 of infusion and subsequent courses due to hypotension. One patient experienced blurred vision during course 2 at a dose level of 12 mg/m2/d requiring a 50% dose reduction for course 3. Another patient at 12 mg/m2/d required omission of the infusion on day 3, course 2 and subsequent dose reduction of 50% for course 3 due to hypotension. One patient who entered the study at 12 mg/m2/d required a 50% dose reduction for course 3 due to a hand and foot rash reaction that occurred during course 2. One patient who entered the study at 14.4 mg/m2/d required a 50% dose reduction for course 2 due to neutropenia and did not complete a third course of therapy. Two patients (13% of patients completing three courses of immunocytokine) required dose modifications during course 3. One patient experienced hemorrhagic cystitis on day 2 of course 3 (likely related to prior cyclophosphamide treatment) at 2 mg/m2/d necessitating a 50% dose reduction for the fourth course. The same patient who had the hand and foot rash, also became hypotensive during course 3 at a dose of 6 mg/m2/d, requiring further dose reduction by 50% on day 3 of course 3. All of these toxicities were reversible upon completion of protocol therapy.
MTD determination in the first course of treatment
The MTD for this study was defined in the protocol as the highest dose level at which no more than two of six treated patients had DLT in the first course of treatment. All episodes of DLT are shown in . Three additional patients were treated at 2 mg/m2/d because one patient had neutropenia. Dose escalation proceeded for each of the next five dose levels; three evaluable patients were treated in each without DLT during course 1. This included the first three patients in course 1 at the dose of 12 mg/m2/d. One of three had DLT (neutropenia and leukopenia) at 14.4 mg/m2/d. Traditionally, three more patients would have been enrolled at 14.4 mg/m2/d as course 1 was tolerated well by two of three patients at that dose level. However, some patients were showing IL-2–related hypotension in courses 2 to 4 at lower doses (). Of the four patients with grade 3 or 4 hypotension, three of these developed hypotension only in the latter courses (). This hypotension in courses 2 to 4, while significant, did not meet DLT criteria for the MTD determination since it occurred after course 1. Thus, the decision was made to enroll the next three patients at 12 mg/m2/d to further evaluate late toxicities with hu14.18-IL2 at this lower dose. One of these three patients had a DLT during the first course. This was an anaphylactic reaction requiring epinephrine, benadryl, dexamethasone, nebulized albuterol, and supplemental oxygen. This patient had no history of prior biologic/mAb therapy before enrollment onto this study. Thus, five of six patients tolerated the first course of therapy at this dose. Even so, one of these five patients did have significant DLT of hypotension during course 2. Therefore, 12 mg/m2/d was determined to be the appropriate phase II dose, and it was designated as the MTD for this study acknowledging that the protocol defined MTD of this agent may not have been reached in the phase I study. No additional patients were entered.
DLTs observed during treatment with hu14.18-IL2
A total of 28 patients were treated on this study. Fifty-four percent (15 of 28) had stable disease for two or more courses of hu14.18-IL2 therapy. Those patients with stable disease following four courses of therapy with hu14.18-IL2 went on to receive a variety of therapeutic interventions following completion of immunocytokine treatment. With a median follow-up of 20 months, 57% (16 of 28) of all patients enrolled on this study were deceased. All deaths were related to his/her disease. There were no deaths reported while on protocol therapy or within one month following completion of protocol therapy.
Although this phase I study was not designed to evaluate overall tumor response to therapy, all patients were monitored for antitumor activity of hu14.18-IL2. There were no measurable complete or partial responses; however, one patient did show evidence of antitumor activity while receiving immunocytokine.
This patient was an 8-year-old female who at the time of study entry had been heavily pretreated with systemic chemotherapy, radiation, and surgery and had undergone prior stem cell transplant with residual disease shown at multiple sites, seen only by MIBG scan. At the conclusion of 5 months of 13-cis
-retinoic acid treatment, this patient continued to have multiple sites of disease detectable only by MIBG. Because of persistent MIBG positive disease, she then received six cycles of Fenretinide (800 mg/m2
daily × 7 days, followed by 14 days of rest) with minimal change in disease status, as detected by MIBG. She then enrolled in this study and received the allowed 4 courses of hu14.18-IL2 at the lowest dose level (2 mg/m2
/d) with persistent MIBG detectable disease noted at the same multiple sites following the fourth course of treatment. One month after completing immunocytokine therapy, this patient was restarted on fenretinide at 800 mg/m2
daily × 7 days. One week after restarting the fenretinide, an MIBG scan was initially read as normal and reevaluated to show a minimal single parietal skull lesion, consistent with improvement. Based on the possibility that this may have been a delayed clinical response related to the recently completed hu14.18-IL2, approval was granted for two more cycles of hu14.18-IL2 at the same 2 mg/m2
/d dose. At the conclusion of the sixth course of hu14.18-IL2, radiographic evidence of disease remained essentially unchanged. This patient was then restarted on fenretinide 1 week following the aforementioned MIBG scan. Repeat imaging 3 months later showed no evidence of disease. This patient has had several MIBG studies done with no radiographic evidence of disease at this time, now 2.5 years after completing hu14.18-IL2 therapy. It should be noted, however, that the disappearance of MIBG is possible in patients with neuroblastoma even without intervention. Late disappearance of MIBG can be observed due to loss of MIBG avidity by the tumor, as well as technical factors that can modify MIBG uptake (21
). At the time of study entry, this patient’s bone marrow showed one neuroblastoma cell per 100,000 cells by immunocytochemistry. At the conclusion of four courses of hu14.18-IL2, this patient’s bone marrow had three neuroblastoma cells per 100,000 cells with occasional ganglioneuroma cells noted. This would suggest stability of bone marrow disease based on immunocytochemistry after four courses of immunocytokine therapy. Repeat bone marrow since then have shown occasional ganglioneuroma cells, of uncertain significance, and a repeat immunocytochemistry assay detected no neuroblastoma.
In addition, two patients with bulky disease as well as metastatic sites, including the bone marrow, showed evidence of decreasing bone marrow disease. One was detected by repeated bone marrow histology, and one was determined by a change in immunocytochemistry. Despite these changes in bone marrow evaluations, these two patients showed concurrent progressive bulky disease at other sites. Although this may represent microscopic antitumor activity of hu14.18-IL2, it could also reflect nonuniform distribution of neuroblastoma in the bone marrow and sampling error involved in obtaining bone marrow specimens. Therefore, despite the absence of measurable antitumor responses, there is suggestion of antitumor activity of immunocytokine in these three patients.
The pharmacokinetic variables (AUC, peak concentration, half-life, and clearance) for days 1 and 3 of course 1 are shown in . There was a significant decrease in AUC, peak concentration and half-life from days 1 to 3 during courses 1 and 2 (P < 0.03 for each variable). For course 3, this comparison showed significance of half-life only, and in course 4, there was no noted significance. This lack of statistical significance in the last two courses may be explained by decreasing statistical power as the number of patients who received a third and fourth course decreased. When comparing dose of immunocytokine to these variables on days 1 and 3, there was dose dependence for AUC of hu14.18-IL2 on days 1 and 3 and for the peak concentration of immunocytokine value for each of those days during course 1(P < 0.001). There was no dose dependence with clearance. Comparing pharmacokinetic variables per course on days 1 and 3, and evaluating for dose proportionality, all variables in each course showed dose proportionality. The median half-life of hu14.18-IL2, as measured on course 1, day 1 was 3.1 hours.
Pharmacokinetic variables for course 1
Changes in peripheral blood lymphocyte counts have been long noted as a dose-dependent immune modification observed during IL-2 therapy (22
). As expected, there was a peripheral lymphopenia noted on days 2 to 4 of each course during the days of the hu14.18-IL2 treatment. This was followed by a rebound lymphocytosis seen on day 8 (), which persisted through day 22. This pattern was shown in each course of immunocytokine treatment. We reported similar patterns of blood lymphocyte counts when immunocytokine was used to treat adults with metastatic melanoma (7
). The noted lymphopenia followed by lymphocytosis was shown in all courses of immunocytokine treatment; however, in courses 2 to 4, the differences were not statistically significant. This may be due to fewer numbers of patients in those courses resulting in less statistical power. There was a relationship between the dose of hu14.18-IL2 administered and the increase in lymphocyte count from baseline (day 0) to day 8 in course 1 (P
= 0.002, ). There was also a dose effect noted in course 2 (P
= 0.006). When comparing lymphocyte counts with peak concentration of hu14.18-IL2, there is a positive correlation between the change in lymphocyte counts from days 0 to 8 compared with peak immunocytokine concentrations on days 1 and 3 (P
= 0.001 and P
= 0.0006, respectively; ).
Lymphocyte counts. Columns, mean lymphocyte counts of all 28 patients in all four courses of immunocytokine therapy based on blood samples before treatment on the indicated days; bars, SE.
Fig. 2 Correlation of boost in lymphocyte counts on day 8 (lymphocyte count on day 8 lymphocyte count on day 0) of course 1 and dose level. The boost in lymphocyte count on day 8 shows a positive correlation with the dose of immunocytokine given (P = 0.002). (more ...)
Fig. 3 Correlation of peak concentration of hu14.18-IL2 detected on day 1 and on day 3 with the boost in lymphocyte count detected on day 8 (value for day 8 value for day 0) for course 1. Spearman’s rank correlation analysis was done to correlate a change (more ...)
Immune activation/modulation was also determined based on soluble IL-2 receptor (sIL2R) levels in patient serum (). There is evidence to suggest immune activation after immunocytokine infusion as the level of sIL2R was significantly lower on day 1, course 1, compared with days 2 to 8 (P < 0.001). Although the baseline level of sIL2R of courses 1 and 2 seem to be similar, the level on day 1, course 2 is higher than day 1, course 1(P = 0.02), indicating a statistically significant higher baseline level of sIL2R for course 2 compared with course 1, due to the immune activation induced in course 1. Similarly, the sIL2R AUC values for courses 2, 3, and 4 were statistically greater than that of course 1 (P = 0.002, 0.04, and 0.06, respectively) with an overall course effect showing higher sIL2R AUC values with subsequent courses (P = 0.008). For courses 1 and 2, the sIL2R values also increased with increasing dose of hu14.18-IL2 administered (P = 0.002). The sIL2R AUC values in course 1 correlated directly with the peak level of hu14.18-IL2 in the serum on day 1 of course 1 (P = 0.009; ). Therefore, a higher sIL2R AUC was associated with higher maximum concentrations of immunocytokine.
sIL2R levels on various days of each course of treatment. Columns, average serum sIL2R levels obtained for serum samples from all patients for each of the designated courses; bars, SE.
Fig. 5 Correlation between sIL2 receptor levels and peak concentration of immunocytokine. AUC of serum sIL2R levels measured from days 0 to 22, during course 1 was computed using the trapezoid rule for each subject. Spearman’s rank correlation coefficient (more ...)
Antibody responses against the hu14.18-IL2 immunocytokine
Serum samples from multiple times for each course were evaluated at a low dilution factor of 1:5 for the development of antibody directed at hu14.18-IL2. Based on the structure of the immunocytokine, patients could generate antibody against the idiotype of the immunocytokine, designated anti-idiotype antibody, or against the Fc-IL2 portion of the immunocytokine, designated as anti-Fc-IL2 antibodies. The anti-idiotype antibody and anti-Fc-IL2 antibody were detected in inhibition ELISAs, where the anti-idiotype in the patient serum inhibited the hu14.18 antibody from binding to an ELISA plate coated with a murine monoclonal anti-idiotypic antibody with specificity for the 14.18 idiotype; the anti-Fc-IL2 in patient serum inhibited the Fc-IL2 fragment of the immunocytokine from binding to a plate coated with rat anti-human IL-2 antibody (see Patients and Methods). Inhibition of >29% was considered to be positive (+; based on variability of control specimens) for both the anti-idiotypic and anti-Fc-IL2 antibody responses. A (++) response was defined as >50% and 40% inhibition in the anti-idiotypic and anti-Fc-IL2 assays, respectively. In this study, >60% of all patients showed evidence of the development of a (++) anti-idiotype antibody in response to immunocytokine therapy. Similarly, ~50% of these patients developed a (++) anti-Fc-IL2 antibody response (), as defined by inhibition of binding to a rat anti-human IL-2 antibody. Furthermore, the incidence of (++) anti-idiotype and anti-Fc-IL2 antibody responses seemed to increase with subsequent courses of treatment. The functional and clinical significance of these anti-immunocytokine antibodies is beyond the scope of this article and will be the subject of a separate report.10
Percentage of patients demonstrating antibody response to hu14.18-IL2 following each course