Abstract
Background
Denileukin diftitox (ONTAK) is a diphtheria/IL-2R fusion protein able to deplete regulatory T cells in peripheral blood. Regulatory T cells in the local immune microenvironment have been shown to be associated with poor prognosis in ovarian cancer. This study examined whether denileukin diftitox (ONTAK) could be safely administered intraperitoneal in patients with advanced refractory ovarian cancer and assessed its effects on regulatory T cells and tumor associated cytokines in ascites and peripheral blood.
Patients and methods
A phase I dose escalation study of intraperitoneal denileukin diftitox (ONTAK) enrolled 10 patients with advanced, refractory ovarian carcinoma at 3 doses (5 ug/kg, 15 ug/kg, and 25 ug/kg). Serial CA-125 measurements assessed clinical response. Regulatory T cells were quantified using RT-PCR and cytokine levels measured by Luminex.
Results
The maximum tolerated dose was 15 ug/kg with a dose limiting toxicity observed in 1 out of 6 patients in the expansion group. The majority of adverse events were transient grades 1–2. One patient treated at the 25 ug/kg dose experienced cytokine storm with prolonged hospitalization. 3 patients had decreases in CA-125 after treatment but none met criteria for partial response. Treatment with denileukin diftitox (ONTAK) decreased regulatory T cells in peripheral blood and ascites. Treated patients did not show any significant changes in IL-8, TGF-β, sIL2Ra in ascites or peripheral blood.
Conclusions
Denileukin diftitox (ONTAK) can be safely administered intraperitoneally to recurrent refractory ovarian cancer patients. Regulatory T cells were reduced in ascites and peripheral blood, but there were no significant changes in cytokine levels.
Keywords: ovarian cancer, denileukin diftitox, regulatory T cells, intraperitoneal immunotherapy
Introduction
Ovarian cancer is immunogenic and infiltration of T lymphocytes is associated with improved prognosis.[1, 2] Despite the importance of the immune response in ovarian cancer, immunotherapies such as checkpoint inhibitors and vaccines to ovarian cancer have yielded only modest clinical responses.[3–6] These limited responses may be due in part to immunosuppressive mechanisms observed in the ovarian cancer tumor microenvironment.[7–10]
Ovarian cancers may evade immune destruction in the peritoneum through a complex network of immunosuppressive factors including cytokines and soluble effector cells thought to contribute to tumor growth, which include active immune suppression by regulatory T cells. CD4+CD25+ regulatory T cells play a role in suppressing immune cells, where they might otherwise recognize and destroy ovarian tumors and they are associated with high death hazard and reduced survival.[7] Regulatory T cells are important in the local immune environment of ovarian cancer and preferentially home to tumors and associated ascites where they suppress production of interferon gamma and interleukin 2 by T cells.[7] Tumor infiltrating lymphocytes from ovarian cancer patients contain increased proportions of regulatory T cells secreting immunosuppressive cytokines like TGF-β. [11] The ratio of regulatory T cells to T helper 17 cells is higher in ovarian cancers than in normal peritoneum[12].
Denileukin diftitox (ONTAK), a diphtheria/IL-2R fusion protein, depletes peripheral blood regulatory T cells in preclinical models of ovarian cancer and when given intravenously to ovarian cancer patients.[13] Because of the importance of the local immune microenvironment in ovarian cancer we hypothesized intraperitoneal (I.P.) denileukin diftitox (ONTAK) administration could reduce regulatory T cells in the tumor microenvironment and boost a productive antitumor immune response at the tumor site. High levels of tumor regulatory T cells in neu-tg mice are associated with immune suppression and depletion of regulatory T cells using denileukin diftitox (ONTAK) could augment tumor antigen-specific immunity and result in tumor regression. [14]
This study examined whether I.P. infusion of denileukin diftitox (ONTAK) was safe and able to reverse immune suppression in the peritoneum by depleting local regulatory T cells. The primary goal of this study was to assess safety and estimate the maximum tolerated dose (MTD) of I.P. administration of denileukin diftitox (ONTAK).
Methods
Patient Population
After informed consent was obtained, patients were enrolled in this trial approved by the University of Washington Human Subjects Division (NCT00357448). All patients had a histological diagnosis of advanced stage epithelial ovarian, primary peritoneal, or fallopian tube carcinoma, refractory to platinum-based chemotherapy, a period of 30 days since last chemotherapy, and previous chemotherapy regimen that included a platinum-based regimen with paclitaxel. Other enrollment criteria include adequate hematologic, renal, and hepatic laboratory parameters, a Performance Status Score (Zubrod/SWOG Scale) ≤ 2, recovery from major infections and/or surgical procedures, and no significant active concurrent medical illness precluding protocol treatment.
Study Design
This was a Phase I, non-randomized, dose escalation study of intraperitoneal (I.P.) denileukin diftitox (ONTAK) administration in patients with advanced, refractory ovarian carcinoma. Three dose levels of denileukin diftitox (ONTAK) were investigated (5 ug/kg, 15 ug/kg, and 25 ug/kg) chosen based on previous safe and effective IV doses.[15] There was a planned enrollment of up to 6 patients/dose level. Inclusion and exclusion criteria, dose escalation schema with DLT definitions, drug information, and drug administration procedures can be found in Supplemental Information. On days 1–3, patients were given I.P. denileukin diftitox (ONTAK). Follow up assessments were conducted on days 8 and 14 of each 14-day treatment cycle.
The primary objective was to estimate the maximum tolerated dose (MTD) of I.P. administration of denileukin diftitox (ONTAK). Toxicities were evaluated based on the Cancer Therapy Evaluation Program’s (CTEP) Common Terminology Criteria for Adverse (CTCAE) v3.0 and monitoring of adverse events was done per Food and Drug Administration (FDA) and National Cancer Institute (NCI) guidelines.
The secondary endpoints were to evaluate the change in number of regulatory T cells in the peritoneum and peripheral blood with administration of denileukin diftitox (ONTAK), assess the clinical impact of denileukin diftitox (ONTAK) on tumor burden through analysis of serial CA-125 measurements, and to assess the level of circulating cytokines (IL-2, IL-6, IL-10, TGF-β2, TNF-α, and sIL2Ra) in the peritoneum and peripheral blood before and after treatment with I.P. denileukin diftitox (ONTAK). Immunologic efficacy of denileukin diftitox (ONTAK) was defined as a 25% reduction in the number of regulatory T cells from baseline in either the peripheral blood or peritoneal cavity.
Evaluation of regulatory T cells
PBMC were isolated from heparinized peripheral blood by density gradient centrifugation.[16] Tumor-associated lymphocytes were isolated from heparinized ascites fluid or I.P. washings using a discontinuous Ficoll gradient.[17] CD4+ CD25+ FOXp3+ regulatory T cell were measured by RT-PCR as previously published.[14]
Evaluation of cytokines
The detection of cytokines IL-2, IL-6, IL-10, TGF-β2, TNF-α, and sIL-2R in both sera samples and ascites T cell supernatant was performed using a multiplex cytokine assay kit (Millipore) and evaluated on a Luminex instrument as previously described.[18]
Evaluation of CA-125 response
Patients had serum CA-125 measured at baseline and monthly for 3 months and 6 months post treatment. Responses were characterized using defined criteria for response and progression in ovarian cancer clinical trials.[19]
Statistical Analysis
Statistical analyses were performed using GraphPad Prism 6.0 (GraphPad Software, Inc., San Diego, CA). The Mann-Whitney test was used to compare differences in cell populations before and after treatment.
Results
Denileukin diftitox (ONTAK) was well tolerated at the 5 ug/kg dose level and the 15 ug/kg dose level had only one patient with dose limiting toxicity after expansion to 6 patients
A total of 10 patients were treated in this Phase 1 trial (Supplemental Figure 1). The majority of patients had stage 3 disease at diagnosis, but there was a wide range of intervals from initial diagnosis to study enrollment and from diagnosis of their recurrence to enrollment. The patients were heavily pretreated, all having had at least 2 prior chemotherapy regimens and as many as 7 (Table 1). The first two dose levels were well tolerated (Table 2). Toxicity for the 5 ug/kg and 15 ug/kg dose levels were primarily transient grade 1 or 2 hypoalbuminemia and abdominal symptoms. No DLTs were observed at the 5 ug/kg dose level. At the 15 ug/kg, dose level, one patient experienced grade 3 gastrointestinal AEs that qualified as DLT and this dose level was expanded to 3 more patients. No DLT were observed in those patients and therefore 1 out of 6 patients at the 15 ug/kg dose level experienced DLT.
Table 1.
Baseline patient characteristics (n = 10).
| Characteristic | Median (range) | No. | % |
|---|---|---|---|
| Number of patients | – | 10 | – |
| Age. years | 56(34–73) | – | – |
| Disease Stage | |||
| III | – | 7 | 70% |
| IV | – | 3 | 30% |
| CA-125 | 342 (32–1055) | – | – |
| Time from diagnosis months | |||
| Intial Diagnosis | 18(10–69) | – | – |
| Recurrent Diagnosis | 8 (2–53) | – | – |
| Time from last chemotherapy, months | 3 (1–4) | – | – |
| Number of previous chemotherapy regimens | 3 (2–7) | – | – |
Table 2.
Adverse events (n = 10).
| Adverse Events | ||||||||
|---|---|---|---|---|---|---|---|---|
| Event | All | Possibly, Probably, or Definitely Related |
||||||
| |
Arm 1 (5 μg/kg) n = 3 |
Arm 2 (15 pg/kg) n = 6 |
Arm 3 (25 pg/kg) n = 1 |
|||||
| No. | % of All AEs | No. | %* | No. | %* | No. | %* | |
| Most Common AEs | ||||||||
| Abdomen pain (NOS) | 29 | 7 | 4 | 1 | 21 | 6 | 0 | 0 |
| Abdominal distension/bloating | 29 | 7 | 5 | 1 | 20 | 6 | 1 | <1 |
| Hypoalbuminemia | 27 | 6 | 6 | 2 | 17 | 5 | 1 | <1 |
| Vomiting | 21 | 5 | 2 | 1 | 15 | 4 | 0 | 0 |
| Fatigue | 19 | 4 | 3 | 1 | 14 | 4 | 0 | 0 |
| Hypokalemia | 19 | 4 | 6 | 2 | 12 | 4 | 0 | 0 |
| Fever (in absence of neutropenia) | 17 | 4 | 6 | 2 | 9 | 3 | 1 | <1 |
| Nausea | 17 | 4 | 3 | 1 | 12 | 4 | 0 | 0 |
| Headache | 17 | 4 | 10 | 3 | 6 | 2 | 0 | 0 |
| Capillary Leak Syndrome | 16 | 4 | 3 | 1 | 11 | 3 | 2 | 1 |
| All AE grading (n = 443) | ||||||||
| 1 | 296 | 67 | 52 | 16 | 157 | 47 | 8 | 2 |
| 2 | 133 | 30 | 15 | 4 | 86 | 26 | 4 | 1 |
| 3 | 13 | 3 | 1 | <1 | 11 | 3 | 0 | 0 |
| 4 | 1 | <1 | 0 | 0 | 0 | 0 | 1 | <1 |
| 5 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
Abbreviation: AE, adverse event.
Of all related AEs.
The third arm at 25 ug/kg, was closed to further enrollment after the first patient had a serious adverse event, grade 4 vascular leak syndrome/cytokine storm, in the first cycle. This patient presented with shortness of breath and tachycardia two days after completing the first IP infusions of denileukin diftitox (ONTAK) and found to have pericardial and pleural effusions. Pulmonary embolism and myocardial infarction were ruled out and respiratory viral swabs were negative. On the third hospital day, the patient developed acute respiratory distress and mental status changes. The patient was intubated, admitted to the ICU, and required pressors for hypotension. The patient underwent placement of bilateral chest tubes and a pericardial window. The patient was ultimately extubated and weaned off pressors after 5 days in the ICU and was discharged after a 24-day hospitalization.
Patients treated at the 5 ug/kg and 15 ug/kg dose levels showed decreased regulatory T cells in peripheral blood and ascites
The mean PBMC Foxp3 expression of regulatory T cells in the peripheral blood at baseline compared to week 8 decreased from 0.1726 (± 0.0442, n=6) to 0.03744 (± 0.01014, n=5) (p=0.0275) over all treatment doses (Figure 1A). This translated to a mean 58.9% reduction for arm 1 and 83.21% reduction for arm 2, thus the secondary objective was met for reduction of regulatory T cells in peripheral blood. A greater than 25% reduction in PBMC Foxp3 expression after 8 weeks of treatment compared to baseline was achieved in 5 patients. The mean reduction after 8 weeks of treatment was 73%. When these data were broken down by dose level, the decrease in PBMC Foxp3 after 8 weeks was not significant at the 5ug/kg dose level (p = 0.1500, Figure 1B), but was significant at the 15 ug/kg dose level (P = 0.0374, Figure 1C).
Figure 1:
Foxp3 mRNA expression showing regulatory T cells (expressed as relative quantitation level of Foxp3 normalized to CD4) populations prior to and after starting denileukin diftitox treatment in (A) peripheral blood mononuclear cells (PBMC) (n= 6 for weeks 0 and 4 and n=5 for week 8), (B) PBMC in arm 1 (5 ug/kg) patients (n=3 for weeks 0 and 4 and n=2 for week 8) (C) PBMC in arm 2 (15 ug/kg) patients (n=3) and (D) ascites (n= 3). The dashed lines indicate the reported mean values of % Foxp3 expression in PBMC and ascites fluid of patients with ovarian cancer [4].
Mean TIL Foxp3 expression of regulatory T cells in ascites at baseline compared to week 8 decreased from 0.1855 (± 0.09448, n=3) to 0.05967 (± 0.03043, n=3) (p= 0.2737). A greater than 25% reduction in Foxp3 expression after 8 weeks of treatment compared to baseline was achieved in 3 patients meeting the secondary objective for reduction of regulatory T cells in ascites. The mean reduction after 8 weeks of treatment was 67% (Figure 1D).
Cytokine levels (pg/ml) of IL-8, TGF-β, sIL2Ra in serum and ascites of patients prior to and after starting denileukin diftitox (ONTAK) treatment showed a wide range of values. Levels of all three cytokines measured 4 and 8 weeks after starting therapy were not significantly different from baseline levels.
One patient at the 15 ug/kg dose levels showed progression by CA-125 criteria as best overall response
Of the 3 patients treated with 5 ug/kg of denileukin diftitox (ONTAK), none showed decreases in serial CA-125 measurements with treatment (Figure 2). Of the 3 patients allocated to the 5 ug/kg dose, one achieved stable disease after 27 days on therapy. Disease stabilization was transient and lasted 29 days with a total follow-up period of 74 days.
Figure 2:
Serial measurements of serum CA-125 (U/mL) in arm 1 (5 ug/kg) and arm 2 (15 ug/kg) prior to and after starting denileukin diftitox treatment. CA-125 responses were categorized as stable disease (SD) and progressive disease (PD) based on Gynecologic Cancer Intergroup (GCIG) definitions. The dashed lines indicate the level to achieve a partial CA-125 response (a 50% reduction in CA-125 from baseline) as well as progressive disease (a greater than or equal to 2 times the nadir value measurement in CA-125) [5]. The shaded region in between these two values represents stable disease.
One of the 4 patients that completed all treatment cycles at the 15 ug/kg dose level showed progression by CA-125 (Figure 2). The mean reduction in CA-125 between pre-treatment and mid-treatment seen at this dose level was 11%. The mean CA-125 reading decreased from 469.3 (± 254.6, n=4) before treatment to 416.8 (± 220.4, n=4) p value of 0.8812. Although the CA-125 readings did decrease in patients treated with 15 ug/kg, none met criteria for response nor were statistically significant.[5] No patients met criteria for response after a mean of 27.5 days on therapy (range 7–48). Disease stabilization was longer for the 15 ug/kg dose level and was maintained for a mean duration of 62.3 days (range 35–90) with an average follow-up period of 89.8 days (range 63–103).
Discussion
Denileukin diftitox (ONTAK) can be delivered intraperitoneal at the 15 ug/kg dose with an acceptable toxicity profile to heavily pre-treated, recurrent refractory ovarian cancer patients. Regulatory T cells were reduced with denileukin diftitox (ONTAK) treatment in both the peripheral circulation and ascites at both the 5 ug/kg and 15 ug/kg dose levels meet predetermined immunologic efficacy criteria in the secondary objectives. This suggests immunotherapies delivered locally may exert effects in the periphery and that there could be trafficking of T cells between these immune compartments in response to local and peripheral immune forces. However, we acknowledge a lower proportion of patients had ascites available for collection and analysis, limiting the interpretation of these measurements.
Patients treated with 5 and 15 ug/kg of denileukin diftitox (ONTAK) showed disease stabilization based on CA-125, although no patients achieved a complete or partial response. However, the mechanisms of immune therapies may make CA-125 an imperfect biochemical response marker in ovarian cancer.[19–21] Inflammatory conditions in the peritoneum that are unrelated to cancer progression are known to elevate CA-125 levels. Rustin’s CA-125 criteria for response in ovarian cancer has been validated for cytotoxic chemotherapies, but not immunotherapies.[19] Therefore, there may be limitations to the application of CA-125 for response in immune therapy trials. More accurate markers must be investigated as surrogates for future studies, especially given the competing systems proposed for radiographic immunotherapy response criteria and the limitations of radiographic imaging modalities for detecting ovarian cancer.[22]
Strategies to therapeutically deplete regulatory T cells have been studied in a number of malignancies, but the strength of the association with clinical responses and ability to synergize with immune based therapies is still unclear. In colorectal cancer low dose cyclophosphamide reduces proportional and absolute regulatory T cells, boosting boost in tumor specific T cell responses and prolongs PFS.[23] Stimulation of the CD27 pathway with varlilumab in patients with advanced solid tumors has shown depletion of regulatory T cells with a partial response observed in metastatic renal cell carcinoma in addition to durable disease stability.[24] Depletion of regulatory T cells has been shown to promote tumor-specific immune responses in ovarian cancer.[7] However, there are significant differences seen in regulatory T cells in peripheral blood, ascites, and the tumor.[25] Peripheral blood regulatory T cell frequency is elevated in ovarian cancer compared to healthy controls.[26] CD4+CD25+Foxp3+ T cells make up a greater percent of total cell populations in TIL and ascites when compared to peripheral blood, but may have more immunosuppressive function in TIL.[25, 27] Ascites show a greater proportion of activated/suppressive regulatory T cells when compared to peripheral blood.[28] Circulating CD4+CD25+FoxP3+ regulatory T cells are associated with disease progression and thus might be considered a predictor of poor survival in patients with cancers such as hepatocellular carcinoma.[29, 30] Regulatory T cell biology may be even more complex in recurrent ovarian cancer, where chronic inflammation developed over multiple lines of chemotherapy may increase the significance of IL-17+CD4+ Th17 cells such as have been observed to contribute to the pathogenesis of ulcerative colitis.[12, 31]
Intraperitoneal delivery of other immunostimulatory agents have been studied in ovarian cancer. Immune checkpoint blockade has been administered intraperitoneal with and without chemotherapy in an effort to improve the modest response rates seen with intravenous therapy with these agents.[32, 33] Weekly infusions of IL-2 showed associations between CD3 counts and activated CD8 T cells with survival.[34] IL-12 plasmid with and without chemotherapy increases interferon-gamma and TNF-alpha in peritoneal fluid after therapy.[35–37] Catumaxomab is thought to be able to destroy tumor cells in the peritoneal cavity responsible for ascites in ovarian cancer through binding CD3 and EpCAM and decreases ascites related symptoms and also induces interferon-gamma and IL-2 production.[38, 39] Patients treated with denileukin diftitox (ONTAK) did not show any increase or decreases in endogenous immunity through analysis of cytokines, however, only 3 patients produced enough ascites to analyze cytokine levels. Pre-existing T cell responses in the peritoneal cavity may influence the production of ascites and modulating these factors in ovarian cancer has been attempted to treat malignant ascites.
Whether I.P. administration of denileukin diftitox (ONTAK) or any other immunotherapy will represent an advantage in recurrent ovarian cancer over less cumbersome and invasive routes either as monotherapy or in combination with other agents remains an open question. The formulation of denileukin diftitox (ONTAK) used in this study was voluntarily taken off the market due to manufacturing difficulties. A new formulation of denileukin diftitox (Lymphir) has been approved for relapsed/refractory cutaneous, T-cell lymphoma. It has greater bioactivity and is considered a new drug by the FDA, so reconsideration of the IP route may require new studies.[40] Biopsies of intraperitoneal tumors undergoing immunotherapy could provide a more direct assessment of the tumor microenvironment than what could be gleaned from ascites. Closer interrogation and improved understanding of the local immune response in ovarian cancer may yield immune biomarkers to identify which patients may benefit from these local immunotherapy approaches.
Supplementary Material
Supplemental Figure 1: Enrollment, dose allocation, follow-up, and analysis
Highlights.
Ovarian cancer has an immunosuppressive tumor microenvironment that has proven resistant to many immunotherapies.
Denileukin diftitox can be safely given intraperitoneally to treatment refractory ovarian cancer patients.
Intraperitoneal denileukin diftitox reduces regulatory T cells both locally, in ascites, and in the peripheral circulation.
Intraperitoneal immunotherapy is feasible and may decrease immunosuppression in the tumor microenvironment.
The effects of local administration should be considered as novel immunotherapies are developed for ovarian cancer.
Acknowledgements
This research study was supported by pilot project funding from the National Institutes of Health grant NCRR M01 RR 00037, and a National Cancer Institute training grant award to Dr. Salazar (K23 CA10069-01).
Footnotes
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Clinical Trial Registration: ClinicalTrials.gov # NCT00357448
Competing interests statement
JBL receives research funding from Merck, AstraZeneca, Precigen, ArsenalBio, Volastra, Nurix, Laekna and Aminex Therapeutics through his institution and is a consultant for Verismo Therapeutics. WRG is a consultant for Corea Therapeutics, Puma Therapeutics, and AstraZeneca. ALC declares associations with Actuate, Amgen, Seagen/Pfizer, Mirati, NuCana, and AstraZeneca. MLD has grant funding from Precigen, Veanna, Bavarian Nordic, and Aston Sci. MLD also holds shares in Epithany and is an inventor on patents held by the University of Washington.
The authors declare there are no other competing interests.
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Associated Data
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Supplementary Materials
Supplemental Figure 1: Enrollment, dose allocation, follow-up, and analysis