ORIGINAL RESEARCH
Phase I trial of systemic intravenous infusion of interleukin- 13-Pseudomonas exotoxin in patients with metastatic adrenocortical carcinoma
Yi Liu-Chittenden1, Meenu Jain1, Parag Kumar2, Dhaval Patel1, Rachel Aufforth1, Vladimir Neychev1, Samira Sadowski1, Sudheer K. Gara1, Bharat H. Joshi3, Candice Cottle-Delisle1, Roxanne Merkel1, Lily Yang1, Markku Miettinen4, Raj K. Puri3 & Electron Kebebew1
1Endocrine Oncology Branch, National Cancer Institute, National Institutes of Health, Bethesda, Maryland
2Clinical Pharmacokinetics Research Laboratory, Clinical Center Pharmacy Department, National Institutes of Health, Bethesda, Maryland 3Tumor Vaccines and Biotechnology Branch, Division of Cellular and Gene Therapies, Center for Biologics Evaluation and Research, Food and Drug Administration, Silver Spring, Maryland
4Laboratory of Pathology, National Cancer Institute, National Institutes of Health, Bethesda, Maryland
Keywords
IL-13-PE, maximum-tolerated dose, metastatic adrenocortical carcinoma, pharmacokinetics, Phase I, systemic administration
Correspondence
Electron Kebebew, Endocrine Oncology Branch, National Cancer Institute, National Institutes of Health, 10 Center Drive, Room 4-5952, Bethesda, MD 20892. Tel: 301-496-5049; Fax: 301-402-1788; E-mail: kebebewe@mail.nih.gov
Funding Information
This research was supported by the intramural research program of the Center for Cancer Research, National Cancer Institute, National Institutes of Health (grant # 1 ZIA BC011286 05).
Received: 12 January 2015; Revised: 14 February 2015; Accepted: 22 February 2015
Cancer Medicine 2015, 4(7):1060-1068
[Correction added on 22 June 2015, after first online publication: There has been a change in the reference list and reference 3 onwards have been re-numbered in this version.]
Abstract
Adrenocortical carcinoma (ACC) is a rare but lethal malignancy without effec- tive current therapy for metastatic disease. IL-13-PE is a recombinant cytotoxin consisting of human interleukin-13 (IL-13) and a truncated form of Pseudomo- nas exotoxin A (PE). The main objectives of this Phase I dose-escalation trial were to assess the maximum-tolerated dose (MTD), safety, and pharmacokinet- ics (PK) of IL-13-PE in patients with metastatic ACC. Eligible patients had con- firmed IL-13 receptor alpha 2 (IL-13Rx2) expressions in their tumors. IL-13-PE at dose of 1-2 µg/kg was administered intravenously (IV) on day 1, 3, and 5 in a 4-week cycle. Six patients received 1 µg/kg and two patients received 2 µg/kg of IL-13-PE. Dose-limiting toxicity was observed at 2 ug/kg, at which patients exhibited thrombocytopenia and renal insufficiency without requiring dialysis. PK analysis demonstrated that at MTD, the mean maximum serum concentra- tion (Cmax) of IL-13-PE was 21.0 ng/ml, and the terminal half-life of IL-13-PE was 30-39 min. Two (25%) of the eight patients had baseline neutralizing anti- bodies against PE. Three (75%) of the remaining four tested patients developed neutralizing antibodies against IL-13-PE within 14-28 days of initial treatment. Of the five patients treated at MTD and assessed for response, one patient had stable disease for 5.5 months before disease progression; the others progressed within 1-2 months. In conclusion, systemic IV administration of IL-13-PE is safe at 1 µg/kg. All tested patients developed high levels of neutralizing antibod- ies during IL-13-PE treatment. Use of strategies for immunodepletion before IL-13-PE treatment should be considered in future trials.
doi: 10.1002/cam4.449
Introduction
Adrenocortical carcinoma (ACC) is a rare malignancy with an incidence of 0.7-2.0 per million people per year
[1-3]. It has an overall 5-year relative survival rate of 32- 42%, and a median survival of 17-32 months [1, 4]. The treatment of choice for a localized primary or recurrent ACC is surgical resection. However, patients with recur-
rent or metastatic disease are rarely curable by surgery alone. Other therapeutic options such as systemic chemo- therapy and locoregional radiotherapy have limited impact on survival [5]. Thus, identifying new therapeutic targets and strategies are of great importance to the treat- ment of this malignancy.
Interleukin-13 receptor x2 (IL-13Rx2) is a high-affinity receptor for the Th2-derived cytokine interleukin-13 (IL- 13), and is overexpressed in several types of cancers as compared to low or absent expression in embryonic cells and normal tissues [6-11]. The high-affinity binding of IL-13 to IL-13Rx2 signals through a STAT-6-independent AP-1-dependent pathway, which leads to increased trans- forming growth factor-beta (TGF-B) activity [12]. We previously reported that IL-13Rx2 was significantly over- expressed in ACCs as compared to normal adrenocortical tissues and benign adrenocortical tumors [13]. Thus, IL- 13Rx2 represents a promising therapeutic target for ACC and other solid malignancies such as pancreatic adenocar- cinoma and hepatocellular carcinoma [14, 15].
IL-13-PE is a chimeric fusion protein consisting of human IL-13 and a truncated form of Pseudomonas exo- toxin A (cintredekin besudotox, hIL13-PE38QQR) [16, 17]. Previous studies have shown that IL-13-PE can bind to IL-13Rx2 positive tumor cells and is highly cytotoxic to these cells in both in vitro and in vivo models of multi- ple malignancies [16, 18, 19]. It has been demonstrated that IL-13Rx2 positive ACC cell lines were sensitive to IL- 13-PE as well [13]. Moreover, treatment with IL-13-PE caused tumor regression and prolonged survival in a mouse xenograft model of ACC [13]. Several Phase I and II clinical trials and one Phase III clinical trial have been performed to evaluate the safety, tolerability, and efficacy of IL-13-PE using regional delivery of the agent for intra- cranial malignancies [20-24]. Here, we report the first Phase I study of systemic intravenous (IV) administration of IL-13-PE in patients with metastatic ACC.
Materials and Methods
Eligibility
Patients with metastatic ACC who failed standard treat- ments were enrolled. The main eligibility criteria included: age ≥18 years; pathological confirmation of positive IL-13Rx2 expression in ≥30% of the tumor cells by immunohistochemistry (IHC); measurable disease by response evaluation criteria in solid tumors (RECIST v1.1) at presentation; last dose of chemotherapy or last radiotherapy treatment more than 4 weeks prior to start- ing IL-13-PE treatment; prior or current mitotane therapy was allowed if patients were on the therapy to control hy- percortisolemia, tolerating their dose and did not have a
tumor response to treatment; no currently active central nervous system metastasis; Eastern Cooperative Oncology Group (ECOG) performance status at 0-2; good organ function. All patients provided written informed consent. The study (Clinicaltrials.gov: NCT01832974) was approved by our Institutional Review Board and con- ducted in accordance with Helsinki Declaration and good clinical practice guidelines.
Study design
This was an open-label Phase I study to assess the maxi- mum-tolerated dose (MTD) of IL-13-PE. The dose-escala- tion strategy involved three cohorts: six patients to receive 1 µg/kg IL-13-PE, three to six patients to receive 2 µg/kg, and three to six patients to receive 3 µg/kg (Table S1). Treatments for all dose levels were intrave- nously administered over a 1-h infusion on days 1, 3, and 5 of the first week in each 4-week cycle. Patients were planned to receive four cycles of treatment, but additional cycles were allowed if they had stable disease or partial/ complete response. In the following text, detailed treat- ment date will be referred to as C_D_, with C for Cycle and D for Day; specific patient will be referred to as Pt ._.
At each dose, only two patients were on treatment until the first cycle was completed. Additional patients at the same dose were enrolled only when no dose-limiting tox- icity (DLT) was observed in those patients. Escalation to the next higher dose was permitted if no more than 1/6 of the previous cohort experienced DLT or the first three patients in subsequent cohorts did not experience DLT. MTD was defined as the highest dose that induces DLT in less than two patients in a cohort of six patients. Adverse events (AEs) were graded according to the Com- mon Terminology Criteria for Adverse Events (CTCAE version 4.0). No intrapatient dose escalation or dose reductions were allowed.
Evaluation
Blood chemistry and urinalysis were tested within 24 h prior to each treatment and daily during week 1 and then weekly (±2 days). Toxicity evaluation, vital signs, ECOG status, and physical examination were completed within 24 h of each treatment. CT scans of the chest, abdomen and pelvis were used to evaluate the response using RE- CIST criteria v1.1.
Pharmaceutical information
IL-13-PE was produced in Escherichia coli as described previously under clinical grade drug manufacturing by In- sys Therapeutics (Chandler, AZ) [17].
Pharmacokinetics
Blood samples from patients were collected at 30 min before infusion, and at 0, 5, 15, 30, 60, 90, 120, 180, 240 min, and 24 h after infusion completion on C1D1, C1D3, and C2D1 if treatment was continued. Serum sam- ples were obtained by centrifuging the blood sample tubes at 1940 g for 10 min at 4℃. Serum IL-13-PE concentration was measured in duplicates by enzyme-linked immunosor- bent assay (ELISA) using Quantikine Human IL-13 Immu- noassay kit from R&D systems, Minneapolis, MN. Purified IL-13-PE was used to produce the standard curve. The lower limit of quantification for this assay was 1.7 ng/mL.
IL-13-PE serum concentration data were analyzed to cal- culate pharmacokinetics (PK) parameters of apparent elim- ination half-life (tv), area under the concentration-time curve from time 0 to the last time point (AUClast) and extrapolated to infinity (AUC0-0), clearance (CL), and vol- ume of distribution (Vz) via noncompartmental methods using Phoenix WinNonlin Version 6.4 (Pharsight Corp. Mountainview, CA). AUC was computed using the linear (up)/logarithmic (down) trapezoidal rule with a minimum of four quantifiable concentrations. The only observed val- ues included the Cmax and the corresponding peak times (Tmax). Dose-proportionality of AUC and Cmax PK param- eters was assessed by determining the geometric mean ratio (GMR) of the two different dose levels.
Anti-IL-13-PE neutralizing antibody detection
A noncommercial, nonisotopic assay was used to detect neutralizing antibody against IL-13-PE. One thousand U251 cells (Human glioma cell line positive for IL-13Rx2 expression) in 100 uL of complete RPMI 1640 medium were plated on 96-well black-wall clear-bottom plates and allowed to attach in a 37°℃ incubator with 5% CO2 over- night. Medium composition was as described in Ou, W., et al., 2012 [25]. On the second day, 50 uL serially diluted patient serum was added in quadruplicates into the U251 cells-coated wells. After 2-h incubation in the cell culture incubator, 50 uL of purified IL-13-PE at 1 ng/ml was added to each well. The plates were then returned to the incubator. Four days later, 20 µL of house-made resazurin was added to each well. Fluorescence of the plates was mea- sured using SpectraMax M5 (VWR Corporate, Altlanta, GA) plate reader (Molecular Devices, Sunnyvale, CA) after 4 h (544 nm for excitation and 590 nm for emission).
Immunohistochemistry
IHC staining for IL-13Rx2 was performed with Dako Autostainer (Carpinteria, CA). Primary antibody used was
goat anti-IL-13Rx2 antibody (R&D systems, Minneapolis, MN, 1:1000). The signals were detected by ImmPRESS HRP anti-goat Ig (peroxidase) polymer detection kit (Vector Laboratories, Burlingame, CA), developed by DAB+ (Dako, Carpinteria, CA), and followed by a light hematoxylin counterstain. The staining was semiquantita- tively assessed for intensity and percent of positive cells. Negative, weakly positive, and strongly positive controls, selected based on RNA expression data, were included in each experiment. The slides were scanned under an Olympus light microscope (Nikon, Tokyo, Japan) and images were acquired at 20x and 40x magnifications.
Results
Patient characteristics and treatment dose
Eight patients were enrolled in this study (N = 6 at dose 1 µg/kg, N = 2 at dose 2 µg/kg) and were evaluable for toxicity. The clinical characteristics of the patients enrolled are summarized in Table 1. All patients had stage IV ACC with metastasis to lung (100%), liver (50%) and bone (12.5%). Seven (87.5%) patients had previous oper- ations, two patients (25%) had radiotherapies and all eight (100%) patients had no response to prior systemic chemotherapies. All patients had positive IL-13Rx2 expression in ≥30% of the tumor cells as determined by IHC (Fig. 1).
Adverse events
To determine the safety of IL-13-PE, all AEs that were potentially related to the therapy were examined. Of the six patients treated with 1 µg/kg IL-13-PE, the most com- mon AEs included Grade 1 or 2 anemia, proteinuria, increase in alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatinine and fatigue, all of which were seen in three patients. One patient developed Grade 3 hypertension which resolved within 2 days (Table 2). Dose escalation to 2 ug/kg resulted in severe AEs in the first two patients enrolled. Grade 3 anemia was observed in both patients, whereas Grade 3 or 4 pro- teinuria, creatinine increase, acute kidney injury, hyponat- remia, neutropenia, pain, and thrombocytopenia were observed in one of two patients. Both patients recovered from these toxicities with supportive care not requiring hemodialysis. Thus, it was determined that 1 µg/kg was the MTD.
To determine if the DLT was due to a rapid induction of proinflammatory cytokines, we performed a human 30-plex cytokine array using serum samples within 2 days of the initial treatment. The expression levels of 30 com- mon human pro-/anti-inflammatory cytokines were
| Demographic and clinical | N % | |
|---|---|---|
| characteristics | ||
| Sex | ||
| Male | 3 | 37.5 |
| Female | 5 | 62.5 |
| Age at diagnosis, median (range) | 39 (15-64) | |
| Age at enrollment, median (range) | 42 (18-65) | |
| Body weight in kg, median (range) | 82.8 (59.8-125.9) | |
| Performance status | ||
| 0 | 7 | 87.5 |
| 2 | 1 | 12.5 |
| Tumor stage at initial diagnosis IV | 8 | 100 |
| Site of tumor at enrollment | ||
| Primary adrenal gland | 1 | 12.5 |
| Adrenal bed (local recurrence) | 7 | 87.5 |
| Regional intra-abdominal recurrence1 | 1 | 12.5 |
| Lung metastases | 8 | 100 |
| Liver metastases | 4 | 50 |
| Bone metastases | 1 | 12.5 |
| Prior treatment | ||
| Surgery2 | 7 | 87.5 |
| Radiotherapy | 2 | 25 |
| Chemotherapy | 8 | 100 |
| Mitotane monotherapy | 7 | 87.5 |
| Etoposide, doxorubicin, cisplatin, | 8 | 100 |
| and mitotane | ||
| Protease inhibitor3 | 1 | 12.5 |
| Abraxane | 1 | 12.5 |
1A single patient was found to have metastatic disease to the intra- abdominal mesentery.
2Prior surgeries include primary resection of adrenocortical carcinoma, metastectomy, and debulking procedures.
3A single patient underwent treatment with proteasome inhibitors, bortezomib, and carfilzomib.
detected simultaneously within each sample to screen for any candidates whose levels were significantly altered. Among the 30 cytokines tested, eight had expression lev- els that were below the lowest detection limit in all sam- ples; one (RANTES) had an expression level above the highest detection limit in almost all samples. No signifi- cant changes were observed in the expression levels of the remaining 21 cytokines that might have contributed to DLT during the treatment course (Fig. S1).
PK assessment
Serum samples on C1D1 and C1D3 were collected from all eight patients for PK studies, whereas serum samples on C2D1 were collected from Pt.1 and Pt.5. Prior to infu- sion, none of the patients had a quantifiable level of IL- 13-PE in the serum. The time versus serum concentration
| Pt. | IL-13Ra2 Staining Intensity | Percent of Tumor cells Positive | A |
|---|---|---|---|
| 1 | 3+ (strong) | 100% | |
| 2 | 3+ (strong) | 100% | |
| 3 | 3+ (strong) | >60% | |
| 4 | 2+ (moderate) 1-2+ (weak to | 80% | B |
| 5 | moderate) | 30% | |
| 6 | 3+ (strong) | 50-75% | |
| 7 | 3+ (strong) | 60-70% | |
| 8 | 2+ (moderate) | >80% |
Figure 1. IL-13Rx:2 staining intensity and percent positive cells for each enrolled patient with metastatic ACC. (A) Representative hematoxylin and eosin (H&E) staining of patient tumor. (B) Representative IL-13Ro:2 staining of patient tumor. IL, interleukin; ACC, adrenocortical carcinoma.
curves for each patient are shown in Figure 2 and the PK parameters related to each dose and treatment cycle are summarized in Table 3. After infusion, IL-13-PE serum concentrations declined log-linearly without any signifi- cant distribution phase observed. The terminal half-life (t1/2) was measured to be 30-39 min under all conditions tested. On C1D1, the geometric mean (GM) of Cmax for IL-13-PE was 17.5 ng/ml when treated at 1 µg/kg, and 41.2 ng/ml at 2 µg/kg. After multiple dosing in the first cycle of therapy, no significant accumulation was observed, as the GMRs of AUCo oo and Cmax (CID3 vs. C1D1) were 1.23 and 1.20 in the 1 µg/kg dose group and 1.14 and 1.00 in the 2 ug/kg dose group, respectively. In contrast, in the two patients who completed a second cycle of therapy at the 1 ug/kg dose, the GMRs of AUCo-o and Cmax (C2D1 vs. C1D1) were decreased by 73% and 72%, respectively, in Pt.5, but were not decreased in Pt.1 (Fig. 2). The PK of IL-13-PE increased relatively proportionally when the dose was doubled, as the GMRs of AUCo co and Cmax (2 µg/kg vs. 1 µg/kg dose groups) were 2.54 and 2.31, respectively.
Immunogenicity of IL-13-PE
To determine if neutralizing antibodies against PE were present before treatment or generated against IL-13-PE during treatment, serum samples before each treatment cycle plus serum samples from C1D15 (if available) were collected and tested in a nonisotopic cytotoxicity assay. Interestingly, baseline anti-PE antibodies were detected in 2 (25%) of the eight patients at a low titer (50) prior to initiation of C1 treatment (Fig. 3, Table S2). By C1D15, neutralizing antibodies were detected in four (67%) of six
| Common toxicity criteria term | 1 µg/kg (N = 6) | 2 µg/kg (N = 2) | ||
|---|---|---|---|---|
| Grade 1 or 2 N (%) | Grade 3 or 4 N (%) | Grade 1 or 2 N (%) | Grade 3 or 4 N (%) | |
| Anemia | 3 (50) | 0 | 2 (100) | 2 (100) |
| Proteinuria | 3 (50) | 0 | 2 (100) | 1 (50) |
| Alanine aminotransferase increased | 2 (33.3) | 0 | 1 (50) | 0 |
| Aspartate aminotransferase increased | 2 (33.3) | 0 | 1 (50) | 0 |
| Creatinine increased | 2 (33.3) | 0 | 2 (100) | 1 (50) |
| Fatigue | 2 (33.3) | 0 | 1 (50) | 0 |
| Alkaline phosphatase increased | 1 (16.7) | 0 | 0 | 0 |
| Chills | 1 (16.7) | 0 | 0 | 0 |
| Cough | 1 (16.7) | 0 | 0 | 0 |
| Dyspnea | 1 (16.7) | 0 | 0 | 0 |
| Edema limbs | 1 (16.7) | 0 | 1 (50) | 0 |
| Electrocardiogram QT corrected interval prolonged | 1 (16.7) | 0 | 0 | 0 |
| Headache | 1 (16.7) | 0 | 1 (50) | 0 |
| Hyperkalemia | 1 (16.7) | 0 | 0 | 0 |
| Hypertension | 1 (16.7) | 1 (16.7) | 0 | 0 |
| Pericardial effusion | 1 (16.7) | 0 | 0 | 0 |
| Weight gain | 1 (16.7) | 0 | 1 (50) | 0 |
| Acute kidney injury | 0 | 0 | 2 (100) | 1 (50) |
| Blood bilirubin increased | 0 | 0 | 1 (50) | 0 |
| Bruising | 0 | 0 | 1 (50) | 0 |
| Epistaxis | 0 | 0 | 1 (50) | 0 |
| Fall | 0 | 0 | 1 (50) | 0 |
| Hematuria | 0 | 0 | 2 (100) | 0 |
| Hypoalbuminemia | 0 | 0 | 2 (100) | 0 |
| Hypocalcemia | 0 | 0 | 1 (50) | 0 |
| Hypokalemia | 0 | 0 | 2 (100) | 0 |
| Hypomagnesemia | 0 | 0 | 1 (50) | 0 |
| Hyponatremia | 0 | 0 | 1 (50) | 1 (50) |
| Neutropenia | 0 | 0 | 1 (50) | 1 (50) |
| Nausea | 0 | 0 | 1 (50) | 0 |
| Pain | 0 | 0 | 0 | 1 (50) |
| Thrombocytopenia | 0 | 0 | 2 (100) | 1 (50) |
IL-13-PE, interleukin-13-Pseudomonas exotoxin.
patients with available serum samples. These included the two patients who had baseline antibodies and two new patients who developed anti-IL-13-PE antibodies after treatment. At the end of C1 (day 29, immediately prior to C2D1), anti-IL-13-PE antibodies were detected in all three tested patients (100%), one of which developed neutralizing antibodies for the first time. Once the anti- bodies were generated, they were produced rapidly in large quantities. Within 14-28 days of initial detection, the antibody titer was as high as 105.
Treatment response
Although this was a Phase I trial, we were able to evaluate response to treatment in five of the six patients who received the 1 µg/kg dose (Table S2). Pt.2 was taken off study due to disease progression before the end of C1, thus
the response was not measured. Of the five patients assessed, one (20%, Pt.1) had stable disease for 5.5 months and underwent six cycles of treatment, but disease pro- gressed at the end of C6; two patients (40%, Pt.3 and Pt.5) had stable disease for 2 months, but disease progressed at the end of C2; the other two patients (40%, Pt.4 and Pt.6) had progressive disease after C1.
Discussion
IL-13-PE represents a novel therapeutic strategy that spe- cifically targets cells overexpressing IL-13Rx2. Here, we provide the results of the first Phase I clinical trial designed to examine the safety profile and effects of sys- temic IV administration of IL-13-PE in patients with met- astatic ACC overexpressing IL-13Rx2. Based on our study, IV infusion of 1 µg/kg was determined as the
MTD for IL-13-PE. At this dose, the most common adverse effects included low grade anemia, proteinuria, fatigue, and increase in ALT, AST, and creatinine. The first two patients treated with 2 µg/kg of IL-13-PE developed Grade 3 and 4 toxicities most consistent with thrombotic microangiopathy but only required supportive care. Although a kidney biopsy sample was not obtained to determine thrombotic microangiopathy, this toxicity has been previously observed in Phase I trials of other immunotoxins [26]. The self-limited toxicities occurred after completion of the first week of IL-13-PE infusion. The mechanism behind immunotoxin-induced throm- botic microangiopathy is not well understood [26]. Sev- eral mechanisms have been proposed to explain immunotoxin-mediated thrombotic microangiopathy, including toxin-mediated endothelial damage (off-target and targeted effect) and a general proinflammatory response. We analyzed the cytokine profile in all patients before and after IL-13-PE infusion but observed no appreciable change in proinflammatory cytokine profiles before and after IL-13-PE infusion to support the latter hypothesis. As IL-13Rx2 protein expression was negative in normal human tissues as examined by IHC in human tissue array (Fig. S2), and IL-13Rx2 gene expression was also undetected in 14 cases of human activated lymphocytes (with IL-2) and 21 cases of blood lymphocytes (data not shown), it is unlikely that the toxicities exhibited in Pt.7 and Pt.8 were due to off-tar- get effect of IL-13-PE.
As IL-13-PE is a recombinant cytotoxin, which con- tains domain II and III of PE [16], immunogenicity to this cytotoxin may be expected in patients. Neutralizing antibodies present from prior exposure to PE or pro- duced de novo during treatment represents a challenge for therapy using this chimeric cytotoxin. In this study, we observed that antibodies against PE was present at baseline in 2 (25%) of the eight patients. During IL-13- PE treatment, the generation of high titers of antibodies was fairly rapid (within 14-28 days of treatment initia- tion) and prevailed in all patients who remained on treat- ment at MTD. Generation of neutralizing antibody may alter PK and subsequently diminish the efficacy of IL-13- PE. For example, the PK data for C1 and C2 were avail- able for both Pt.1 and Pt.5. In Pt.1 the Cmax and CL were comparable on C1D1 and C2D1. In contrast, the Cmax
achieved in Pt.5 was much lower on C2D1 than on C1D1 (Fig. 2). This would be consistent with the finding that on C2D1, the anti-IL-13-PE antibody titer was much higher in Pt.5 (104) than in Pt.1 (50) (Fig. 3A). High lev- els of existing anti-IL-13-PE antibodies putatively neutral- ized most of the administered IL-13-PE during treatment, thus the concentration of free IL-13-PE measured in serum was significantly lower in Pt.5.
| Dose | Cycle, day | No. of patients | Cmax (ng/ml) | AUClast (min.ng/ml) | AUCO-o (min.ng/ml) | % AUCextrap | t1/2 (min) | Vz (mL) | CL (mL/min) | ||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| GM | CV | GMR | GM | CV | GMR | GM | CV | GMR | GM | CV | GM | CV | GM | CV | GM | CV | |||
| 1 µg/kg | C1D1 | 6 | 17.5 | 33.8 | NA | 1134 | 57 | NA | 1262 | 59 | NA | 9 | 36 | 30 | 78 | 3169 | 26 | 72 | 86 |
| C1D3 | 6 | 21 | 36.6 | 1.2 | 1410 | 51 | 1.24 | 1553 | 49 | 1.23 | 9 | 37 | 34 | 55 | 2917 | 21 | 59 | 82 | |
| C2D1 | 2 | 11.7 | 79.9 | 0.67 | 665 | 92 | 0.59 | 836 | 79 | 0.66 | 15 | 87 | 36 | 15 | 7320 | 78 | 142 | 87 | |
| 2 µg/kg | C1D1 | 2 | 41.2 | 19 | NA | 2963 | 41 | NA | 3089 | 41 | NA | 4 | 9 | 38 | 26 | 1153 | 31 | 21 | 55 |
| C1D3 | 2 | 41.4 | 36.1 | 1 | 3374 | 34 | 1.14 | 3531 | 33 | 1.14 | 4 | 33 | 39 | 12 | 1068 | 37 | 19 | 47 | |
| 1 µg/kg | All days | 6 | 17.9 | 39.4 | NA | 1154 | 56 | NA | 1300 | 55 | NA | 9.8 | 61 | 33 | 60 | 3447 | 73 | 73 | 87 |
| 2 µg/kg | All days | 2 | 41.3 | 23.8 | 2.31 | 3162 | 31 | 2.74 | 3303 | 31 | 2.54 | 4.2 | 21 | 38 | 16 | 1110 | 28 | 20 | 43.2 |
IL-13-PE, interleukin-13-Pseudomonas exotoxin; Cmax, maximum serum concentration; t1/2, terminal half-life; AUClast, area under the curve from time 0 to last quantifiable time point; AUCO-co, area under the curve extrapolated from time 0 to infinity; % AUCextrap, percentage AUC extrapolation; Vz, volume of distribution; CL, clearance; CV, coefficient of variation; GMR, geometric mean ratio; NA, not applicable.
Pt.1
Pt.2
Pt.3
Pt.4
IL-13-PE level (pg/mL)
100,000
C1D1
IL-13-PE level (pg/mL)
100,000
C1D1
C1D3
IL-13-PE level (pg/mL)
100,000
100,000
C1D1
C1D3
C1D1
IL-13-PE level (pg/mL)
C1D3
+ C2D1
C1D3
10,000
10,000
10,000
10,000
1,000
1,000
1,000
1,000
0
60
120
180
240
0
60
120
180
240
0
60
120
180
240
0
60
120
180
240
Time after infusion (min)
Time after infusion (min)
Time after infusion (min)
Time after infusion (min)
Pt.5
Pt.6
Pt.7
Pt.8
IL-13-PE level (pg/mL)
100,000
C1D1
IL-13-PE level (pg/mL)
100,000
100,000
100,000
E
C1D3
C1D1
IL-13-PE level (pg/mL)
C1D1
C1D3
C1D1
IL-13-PE level (pg/mL)
C2D1
C1D3
C1D3
10,000
10,000
10,000
10,000
1,000
1,000
1,000
1,000
0
60
120
180
240
0
60
120
180
240
0
60
120
180
240
0
60
120
180
240
Time after infusion (min)
Time after infusion (min)
Time after infusion (min)
Time after infusion (min)
A
106
Pt. 1
B
8
105
Pt.2
Numbers of patients
7
Tested
-
Pt.3
Antibody positive
6
-
104
Pt.4
5
.
103
Pt.5
4
·
102
Pt.6
Pt.7
3
-
101
Pt.8
2
·
1
·
10º
0
C1D1
C1D15
C2D1
C3D1
C4D1
C5D1
C6D1
C1D1
C1D15
C2D1
C3D1
C4D1
C5D1
C6D1
Cycle of treatment
Cycle of treatment
PK of IL-13-PE showed overall rapid elimination from serum and apparent linear disposition among drug dose levels of 1 and 2 µg/kg, based on evaluation of Cmax and AUC (drug exposure) on each treatment day and cycle. However, due to limited patient numbers, interpatient variability in weight, age, and other factors that may also influence the PK of IL-13-PE, further investigation is needed in future clinical trials involving IL-13-PE. We also do not know if mitotane therapy could have affected the PK data but this is unlikely given that the patients had normal organ function.
Of the five patients treated at MTD and assessed for response, Pt.1 had stable disease for the longest time (5.5 months) before disease progression. Interestingly, Pt.1 had the highest IL-13Rx2 expression level in the tumors and the latest development of neutralizing anti- bodies against IL-13-PE (Figs. 1 and 3A). However, the
limited number of patients makes it difficult to determine whether patient response would correlate with the expres- sion level of IL-13Rx2.
In conclusion, this is the first Phase I trial designed to test the safety and effects of systemic IV administration of IL-13-PE in patients with metastatic ACC. Our study demonstrated that IV infusion of IL-13-PE at dose 1 µg/ kg is safe and tolerated well by patients when adminis- tered every other day for three times during week 1 of a 4-week cycle. However, the generation of neutralizing antibody might hinder the effectiveness of the treatment. In future clinical trials, it would be worthwhile to con- sider strategies for immunodepletion before IL-13-PE treatment to reduce the development of neutralizing anti- bodies to this immunotoxin. Alternatively, other IL-13 fusion proteins with no or low immunogenicity should be developed.
Acknowledgments
This research was supported by the intramural research program of the Center for Cancer Research, National Cancer Institute, National Institutes of Health (grant # 1 ZIA BC011286 05).
Conflict of Interest
None declared.
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Supporting Information
Additional Supporting Information may be found in the online version of this article:
Figure S1. Expression levels of 22 cytokines that were detected in at least one patient using human 30-plex cyto- kine array. Human Cytokine Magnetic 30-Plex Panel (Life technologies, Frederick, MD) was used to quantitatively determine the level of 30 different cytokines in patient serum. Assay plates were run on Luminex100 platform and data analysis was performed using Bio-Plex manager 6.0 (Bio-Rad, Hercules, CA). Pt.1 to Pt.6 received 1 µg/kg
IL-13-PE, whereas Pt.7 and Pt.8 received 2 ug/kg IL-13- PE. Only data within the detection limit of the assay are shown. For RANTES, missing data points were all above the highest detection limit of the assay. For the remaining cytokines, missing data points were all below the lowest detection limit of the assay. No significant changes in cytokine levels were observed that could be associated with DLT. (A) Proinflammatory cytokines. (B) Anti- inflammatory cytokines. Compared to other patients, the cytokine expression profile of Pt.2 was very different. Pt.2 had heavier disease burden than other patients, and the adverse event exhibited in this patient (Grade 3 bone pain, Grade 3 hypokalemia and Grade 2 AST increase) was considered unrelated or unlikely attributed to treat- ment. Thus, the unusual cytokine levels observed in Pt.2 were unlikely to be associated with toxicity.
Figure S2. IL-13Rx2 IHC staining in normal tissue array. FDA998 normal organ tissue array of human, 47 cases/99 cores (US Biomax, Inc., Rockville, MD) was used to stain for IL-13Rx2. (A) Array layout: Ceg, Cerebral gray matter; Cew, Cerebral white and/or gray matter; Ceb, Cerebellum tissue; Adr, Adrenal gland; Ova, Ovary; Pan, Pancreas; Par, Parathyroid gland; Hyp, Hypophysis; Tes, Testis; Thr, Thyroid; Bre, Breast; Spl, Spleen; Ton, Tonsil; Thy, Thymus gland; Bon, Bone; Lun, Lung; Hea, Heart; Eso, Esophagus; Sto, Stomach; Sma, Small intestine; Col, Colon; Liv, Liver; Sal, Salivary gland; Kid, Kidney; Pro, Prostate; Ute, Uterine cervix; Str, Striated muscle; Ski, Skin; Ner, Nerve; Mes, Mesothelium; Eye, Eye; Lar, Lar- ynx. Blue background: malignant tumor; light purple background: cancer adjacent normal tissue; others: nor- mal tissue. (B) IL-13Rx2 staining. All normal tissues were IL-13Rx2-negative as compared to positive control mela- noma sample.
Table S1. Study design.
Table S2. Treatment dosage, IL-13-PE neutralizing anti- body production and response to treatment.