Phase I Study of the Sequential Combination of Interleukin-12 and Interferon Alfa-2b in Advanced Cancer: Evidence for Modulation of Interfer
http://www.100md.com
《临床肿瘤学》
the Divisions of Hematology and Oncology and Surgical Oncology, Human Cancer Genetics, and Center for Biostatistics, Arthur G. James Cancer Hospital and Richard J. Solove Research Institute, The Ohio State University, Columbus, OH
Yale University, New Haven, CT
ABSTRACT
PURPOSE: To evaluate the safety of sequentially administered recombinant (r) human (h) interleukin-12 (IL-12) and interferon alfa-2b (IFN--2b) in patients with advanced cancer and to determine the effects of endogenously produced IFN- on Janus kinase-signal transducer and activator of transcription (Jak-STAT) signal transduction in patient peripheral-blood mononuclear cells (PBMCs).
PATIENTS AND METHODS: Forty-nine patients with metastatic cancer received rhIL-12 on day 1 and IFN--2b on days 2 to 6 of either a 14-day (n = 43) or a 7-day treatment cycle (n = 6). rhIL-12 was initially administered subcutaneously at a dose of 100 ng/kg, whereas IFN--2b was escalated from 1 to 10 million units (MU) in cohorts of three patients (1, 3, 5, 7, or 10 MU). rhIL-12 was subsequently administered intravenously (IV) in escalating doses (100 to 500 ng/kg) to achieve greater IFN- production. Peripheral blood was drawn for measurement of plasma IFN- and the induction of Jak-STAT signal transduction in PBMCs.
RESULTS: No IL-12–or IFN-–related dose-limiting toxicities were observed. There were no responses in 41 assessable patients. Five patients exhibited stable disease lasting 6 months or longer while on therapy. Optimal induction of IFN- by IL-12 occurred after an IV dose of 250 ng/kg. Patient PBMCs exhibited increased levels of STAT1 after IL-12 administration. The peak level of IFN- achieved with IL-12 therapy correlated with the peak level of intracellular STAT1 in patient PBMCs (r = 0.38, P = .021).
CONCLUSION: The combination of rhIL-12 and IFN--2b can be administered sequentially with minimal toxicity. IV administration of rhIL-12 modulates IFN-–induced Jak-STAT signal transduction in patient PBMCs.
INTRODUCTION
Because renal cell carcinomas (RCCs) are relatively chemotherapy-resistant tumors,1 treatment strategies for metastatic RCC and malignant melanomas have focused on immune-based therapies. Interferon alfa (IFN-) produces overall response rates of 15% to 20% in advanced RCC and melanoma. Also, it is the only agent approved for use as adjuvant therapy after surgical resection of high-risk melanoma.2-4 Recently, IFN--2b was chosen to be the control arm in a new, national, phase III trial of the antiangiogenic agent bevacizumab in patients with advanced RCC.5 Thus, IFN- remains an important therapeutic option in several disease settings, and strategies to improve its activity are warranted.
IFN- regulates gene expression in immune effector cells and tumor cells via activation of the Janus kinase–signal transducer and activator of transcription (Jak-STAT) pathway of signal transduction. The binding of IFN- to its receptor activates two receptor-associated Janus family kinases, Jak1 and Tyk2, which subsequently phosphorylate cytoplasmic transcription factors belonging to the STAT family of proteins (STAT1 and STAT2). These ultimately complex with other DNA binding proteins to initiate the transcription of IFN-–responsive genes within immune cells.6-10 In addition, IFN- can exert direct antiproliferative, antiangiogenic, and proapoptotic effects on tumor cells.11,12 Recent studies by our group have shown that the stimulatory effects of IFN- on host immune cells are critically important to its antitumor actions.13,14
Interleukin-12 (IL-12) is produced primarily by monocytes and macrophages15 and stimulates the proliferation and lytic activity of natural killer cells and cytotoxic T cells. In addition, IL-12 induces the secretion of IFN- by both T cells and natural killer cells and promotes the maturation and activation of type 1 helper T cells, thus favoring the development of cellular immunity and immunologic memory over humoral immunity.16 We have previously shown that IFN- produced in response to IL-12 can upregulate the level of Jak-STAT signaling intermediates in both tumor and immune effector cells. Importantly, the upregulation of critical signaling intermediates sensitized host immune cells to lower doses of IFN-, leading to significantly enhanced survival of tumor-bearing mice.17
We hypothesized that recombinant human IL-12 (rhIL-12) pretreatments would sensitize patients to the antitumor effects of low-dose IFN-, leading to increased efficacy and decreased toxicity. We speculated that IL-12 administration might lead to greater endogenous production of IFN- than what could be achieved with a simple injection of recombinant IFN- alone. Therefore, we conducted a phase I trial of IL-12 followed by IFN- in patients with advanced malignancy. The objectives of this trial were to determine the safety and tolerability of this combination cytokine regimen and to determine the effects of IL-12 pretreatment on IFN- signal transduction pathways using flow cytometric assays. The results of this study suggest that modulation of cytokine signal transduction pathways is feasible in the setting of immune-based cancer therapies.
PATIENTS AND METHODS
Objectives and Eligibility
This National Cancer Institute–sponsored phase I trial was conducted at The Ohio State University Comprehensive Cancer Center from August 1998 to January 2001 under Institutional Review Board approval. The primary objective was to determine the maximum-tolerated dose (MTD) of IFN--2b (INTRON A; Schering Corp, Kenilworth, NJ) when preceded by a single dose of IL-12 in patients with advanced malignancy. The secondary objectives were to measure plasma levels of IFN- and to characterize Jak-STAT signal transduction in patient peripheral-blood mononuclear cells (PBMCs) over the course of therapy. Forty-nine patients between the ages of 23 and 84 years were enrolled onto this study. All patients provided informed consent before treatment. This trial was open to patients with advanced cancer of any histology who had a life expectancy of at least 12 weeks. Prior therapy with IFN- was permitted. Patients were required to have a Karnofsky performance status of 70% and adequate hematopoietic, renal, and hepatic function.
Treatment Protocol
Concerns that continuous administration of IL-12 and IFN--2b might be excessively toxic led to the adoption of a biweekly regimen. rhIL-12 was administered on day 1 of each 14-day treatment cycle, followed by subcutaneous (SC) injections of IFN--2b on days 2 through 6 (Fig 1). Injections of rhIL-12 were initially administered SC at a dose of 100 ng/kg (Table 1). After it was determined that SC doses of IL-12 did not induce significant levels of circulating IFN-, the study was amended to permit the IL-12 to be administered as an intravenous (IV) bolus injection (100, 250, or 500 ng/kg). For each dose of IL-12, the IFN--2b component was dose escalated within cohorts of three patients (1, 3, 5, 7, or 10 million units [MU]/d). Individual patients received the same dose of IL-12 and IFN--2b for the duration of the study. All patients received 12 weeks (six cycles) of therapy unless toxicity or disease progression was observed. Disease response evaluation was performed after every 6 cycles, and patients exhibiting a clinical response or stable disease (SD) were offered the option of continuing therapy until disease progression. Late in the study, the protocol was amended to permit patients to receive cytokine therapy on a 7-day cycle. IL-12 was administered IV at 300 ng/kg to all patients in this cohort.
Management of Toxicity
Toxicity was assessed according to the revised National Cancer Institute Common Toxicity Criteria version 2.0. Dose-limiting toxicity (DLT) was defined as any clearly drug-related grade 3 nonhematologic toxicity that did not resolve after a 2-week rest period or that recurred in subsequent treatment cycles or any clearly drug-related grade 4 nonhematologic toxicity. In the absence of DLT, successive cohorts of three patients were entered onto the protocol at increasing doses of IFN--2b. The MTD was defined as one dose level below the dose at which two or more of six patients experienced DLTs.
Procurement of Peripheral Blood
Approximately 8 to 10 mL of peripheral blood was drawn before each injection of cytokine. PBMCs were separated by density gradient centrifugation with Ficoll-Paque (Amersham Pharmacia Biotech, Uppsala, Sweden), and plasma was snap frozen per protocol.18
Quantitation of Plasma IFN-
Patient plasma samples were analyzed in triplicate for IFN- by enzyme-linked immunosorbent assay using commercially available monoclonal antibody (Ab) pairs as previously described.19 Standard curves were prepared for each plate, and, when possible, all plasma samples from a single patient were run on in parallel to minimize interassay variability.
Intracellular Flow Cytometry for STAT1 Levels
Cryopreserved PBMCs were assayed for total STAT1 using a flow cytometric assay as previously described.20,21 Fixed and permeabilized cells were stained with a murine anti-STAT1 Ab (BD Transduction Laboratories, San Diego, CA) followed by secondary Ab (fluorescein isothiocyanate–conjugated goat antimouse; BioSource International, Camarillo, CA) and then analyzed on a Coulter Elite II flow cytometer (Beckman Coulter, Fullerton, CA).
Statistical Analysis
Data were examined for normal distribution. Fisher’s exact test was used to test for differences in IFN- levels after treatment with high- or low-dose IL-12. Student’s t test was used to test whether STAT1 levels were related to IL-12 dose. A nonparametric Spearman rank correlation was used to test whether IFN- levels and intracellular STAT1 were correlated. P < .05 was used to define statistical significance.
RESULTS
Dose Escalation
The characteristics of all patients enrolled onto this study are listed in Table 1. The first 16 patients received an IL-12 dose of 100 ng/kg SC with minimal toxicity; however, this dose did not routinely induce significant production of IFN- (Fig 2). Therefore, subsequent patients (n = 33) received IV doses of IL-12 starting at 100 ng/kg. IL-12 at a dose of 100 ng/kg IV (n = 4) was an ineffective stimulus for IFN- production, and therefore, the dose was increased to 250 ng/kg (n = 17). The IFN--2b component of therapy was dose escalated to 10 MU/d with no DLT. The trial was then amended to permit administration of IL-12 and IFN--2b on a weekly basis. Six patients (patients 38 to 43) received weekly IL-12 at a dose of 300 ng/kg IV followed by IFN--2b SC on days 2 to 6 of each 7-day cycle at a dose of 1 or 3 MU/d (Table 1 and Fig 1). All patients receiving treatments on a 7-day cycle complained of persistent fatigue and/or weakness (grade 1 or 2) by week 4 of therapy. Although these symptoms were manageable, no objective responses were observed, and the trial was amended to return to a 14-day treatment cycle, with the IL-12 dose set at 500 ng/kg IV (n = 6). The MTD was not reached, and the optimal biologic dose of IL-12 (the dose of IL-12 that induced maximal IFN- levels) was determined to be 250 ng/kg IV (see Systemic Levels of IFN-). At this dose, patients routinely tolerated 1 to 10 MU of IFN--2b without difficulty. For all patients, the median number of cycles received was five (range, one to 18 cycles). Forty-five patients received at least one cycle of therapy and were assessable for toxicity, whereas 41 patients completed two cycles of therapy and, therefore, were assessable for response.
Toxicity
No DLTs were encountered during the course of this study. The regimen was well tolerated, and severe adverse events were uncommon. Table 2 reflects the incidence of grade 3 and 4 toxicities across all treatment cycles. There were no deaths on study. Four patients experienced reversible grade 4 nonhematologic toxicities that were deemed unrelated to the study therapy. Patient 30 developed grade 4 dyspnea during the third cycle of therapy (IL-12 250 ng/kg; IFN--2b 5 MU) as a result of bilateral malignant pleural effusions and was taken off study because of disease progression. Patient 32 developed grade 4 myalgia on day 6 of the first cycle of therapy (IL-12 250 ng/kg; IFN--2b 7 MU) that rapidly resolved. The patient subsequently received a total of nine cycles of therapy without recurrence of the myalgia. Patient 37 (IL-12 250 ng/kg; IFN--2b 10 MU) was receiving hydrochlorothiazide for pre-existing lower-extremity edema and developed asymptomatic grade 4 hyponatremia on day 3 of cycle 1 (118 mg/dL). The serum sodium returned to baseline after discontinuation of hydrochlorothiazide. Patient 40 received six cycles of therapy (weekly regimen) without significant toxicity (IL-12 300 mg/kg; IFN--2b 1 MU). However, during cycle 7, this patient developed Pseudomonas pneumonia and sepsis associated with grade 4 hypoxia and hypotension requiring ventilatory support. This patient was removed from the study. In addition, reversible grade 4 neutropenia occurred in patient 31 (IL-12 250 ng/kg; IFN--2b 7 MU) and patient 41 (IL-12 300 mg/kg; IFN--2b 3 MU, weekly cycle), but it was readily reversible and did not prevent these patients from receiving further cycles of therapy. Less severe toxicities that were commonly encountered included fever, chills, and transient fatigue. These toxicities routinely occurred within 2 to 18 hours of IL-12 administration and were most severe after the first dose of IL-12. Pretreatment with acetaminophen (650 mg) or celecoxib (100 to 200 mg) resulted in fewer and less severe reactions and was routinely used in the latter portion of the trial. The most frequent hematologic toxicities were transient lymphopenia and neutropenia, which occurred more often in patients receiving high doses of IFN--2b ( 7 MU).
Efficacy
Forty-one patients completed two cycles of therapy and were assessable for disease response. No objective responses were seen. Five patients completed at least 12 14-day cycles of therapy (ie, 6 months) and were classified as having SD. Patients 38 and 41 received 12 cycles of therapy on a 7-day cycle (3 months total therapy) and were not considered to have SD. All of the SD patients received IV IL-12, and four of the five patients were treated with IL-12 doses of at least 250 ng/kg (Table 3). However, SD was not statistically associated with plasma levels of IFN- (see next section).
Systemic Levels of IFN-
Plasma IFN- levels were evaluated in all patients before each injection of IL-12 or IFN- (n = 49). In the majority of assessable patients (45 of 49 patients; 91%), peak levels of IFN- occurred on day 2 of the treatment cycle (ie, approximately 24 hours after IL-12 administration) and remained elevated over the next few days. Peak IFN- levels were significantly higher in patients receiving IL-12 at 250 or 300 ng/kg IV compared with patients receiving IL-12 100 ng/kg SC (P < .001, Fig 2). An increase in the IL-12 dose to 500 ng/kg did not lead to further enhancement of IFN- production. Also, 95% of patients (21 of 22 patients) receiving the 250 ng/kg or 300 ng/kg doses of IL-12 had elevated levels of IFN- during every treatment cycle (median number of cycles completed = six) compared with just 37% of patients (six of 16 patients; median cycles completed = 5.5) receiving IL-12 at 100 ng/kg SC (P < .05).
Intracellular Levels of STAT1
We used a flow cytometric assay to test whether endogenous production of IFN- after IL-12 administration led to increased expression of Jak-STAT signaling intermediates in PBMCs, as had been observed in a murine model of melanoma.17 STAT1 levels were measured in PBMCs that were procured on days 1 to 6 of cycle 1. Sufficient data for analysis was available for 23 patients. The remaining 26 patients received their SC injections of IFN- at home, and thus, their PBMCs were not available for analysis. Flow cytometric data from two representative patients are shown in Figures 3A and 3B. Further analysis of STAT1 levels (independent of IL-12 dosage) revealed that the levels of STAT1 were markedly upregulated 1 day after IL-12 administration (day 2 value) and continued to increase until reaching a peak on day 4 (P < .01; analysis of variance). Although STAT1 levels began to gradually decline at this point, they typically remained elevated above baseline on days 5 and 6 of each cycle. A post hoc comparison of this data also demonstrated that day 4 STAT1 levels were significantly higher than the levels measured on days 1 or 2 but not significantly different from STAT1 levels observed on days 3, 5, and 6. This profile of increased STAT1 levels after IL-12 administration was evident for all 23 patients. The trend of increasing STAT1 levels from baseline (day 1) to day 6 is illustrated for patients at each dose level of IL-12 (Figs 3C to 3G).
A plot of the maximal increase in STAT1 over baseline for each dose level of IL-12 (cycle 1 values) is presented in Figure 4A. A comparison of the 100 ng/kg SC and 250 and 300 ng/kg IV dose levels revealed no direct relationship between dosage of IL-12 and total STAT1 protein levels (P = .19). Given our hypothesis that the increase in STAT1 within patient PBMCs was mediated by circulating levels of IFN-, the relationship between these two patient characteristics was examined. A plot of maximum STAT1 increase versus maximum plasma IFN- level in week 1 revealed that these two variables were statistically correlated (r = 0.38, P = .021). This finding indicates that endogenously produced IFN- is likely responsible for modulating levels of STAT1 within patient PBMCs (Fig 4B). However, the observed modifications in the Jak-STAT signal transduction pathway were not clinically beneficial.
DISCUSSION
We have demonstrated that administration of IL-12 followed by IFN- was well tolerated in patients with advanced malignancy. Although no responses were observed in 41 assessable patients, five patients had prolonged stabilization of their disease ( 6 months) during the trial. Correlative studies indicated that maximum IFN- levels were achieved on day 2 of each cycle and that IV administration of IL-12 (250 or 300 ng/kg) induced the maximum level of IFN-. Plasma levels of IFN- correlated with the induction of intracellular STAT1 protein within patient PBMCs during cycle 1. These correlative data are the first to demonstrate that IFN-–induced Jak-STAT signal transduction can be modulated in vivo via IL-12 administration. Furthermore, we have demonstrated the utility of a flow cytometric assay for the analysis of signaling in patient cells after cytokine therapy.
We hypothesized that enhanced expression of Jak-STAT signaling components would render patient immune cells more sensitive to subsequent doses of IFN-. Studies of the molecular mechanism of IFN- pretreatment have indicated that its actions are dose dependent and require protein synthesis, which is a process that may require anywhere from 6 to 24 hours.22-24 Therefore, the optimal schema for the administration of IFN- and IFN- would most likely involve pretreatment of patients with IFN- several hours before the administration of IFN-. This schedule has only been studied in a trial conducted by Ernstoff et al,25 who examined the sequential administration of IFN- and IFN- in patents with advanced RCC. They noted complete responses in two patients (n = 36) and partial responses in an additional six patients. Other phase I studies have shown that coadministration of IFN- and IFN- was well tolerated.25-30
IL-12 was chosen as a pretreatment in this study on the basis of its ability to stimulate the sustained endogenous production of IFN-, which is a factor that can markedly enhance STAT1 expression within immune effector cells and tumor cells.6,17,24 Overall, IL-12 administration was an effective stimulus for IFN- production. We hypothesized that the administration of IL-12 would result in greater endogenous production of IFN- than what would typically be achieved via IV or SC injections of recombinant IFN-. In this trial, IFN- levels remained elevated above baseline for at least 48 hours after a single injection of IL-12. The mean elimination half-life of IFN- is reported to be 38 minutes when administered IV and 5.9 hours when administered as an SC injection. Peak plasma concentrations of IFN- occur at 7 hours after SC dosing.31 In a phase I study of recombinant IFN- in cancer patients, Vadhan-Raj et al32 demonstrated that serum IFN- levels returned to baseline within 2 hours after a 6-hour infusion of IFN- 1 mg/m2. Thus, a single injection of IL-12 leads to prolonged elevations in plasma IFN- that are superior to the elevations that can be achieved via administration of the recombinant cytokine.
The induction of IFN- by IL-12 seemed to be variable within each cohort of patients. The IV route of administration was superior to the SC route for the induction of IFN-, but an increase in the IL-12 dose from 300 to 500 ng/kg did not lead to increased production of IFN-. Although limited patient numbers in some of the dose levels prevented comparisons across treatment groups, this observation suggests that the MTD of IL-12 is not necessarily the most biologically active dose. The apparent reduced efficacy of IL-12 at the highest dose tested may be indicative of hormesis, a U-shaped dose-response phenomenon that is characterized by stimulation at low doses and inhibition at higher doses.33
We have previously demonstrated an essential role for STAT1 signal transduction within host immune cells for mediating the antitumor effects of IFN-.13 Therefore, in this trial, it was of particular interest to determine whether STAT1 levels could be modulated in patients with advanced cancer. Levels of intracellular STAT1 protein in patient PBMCs increased dramatically by day 2 of the cycle, reached a peak on day 4, and then declined over the next few days. Importantly, circulating levels of IFN- correlated with the induction of STAT1 protein within patient PBMCs, suggesting that this cytokine was indeed responsible for the changes that had occurred. This effect of IFN- on the Jak-STAT pathway was consistent with what had been previously reported in the context of in vitro studies conducted by our laboratory.17 This effect may partly explain the synergy that is observed when IL-12 is combined with other cytokines34-37 and may represent a functional pathway for optimization of Jak-STAT signal transduction during infectious processes.
IL-12 has been infrequently administered with other cytokines in clinical trials largely because of the fear of synergistic and/or overlapping toxicities. SC administration of low-dose IL-2 enhanced the IFN- response to IL-12 and prevented the IFN- response from declining over time,36 and IL-12 administered twice weekly with IFN- (thrice weekly) resulted in elevated levels of IFN- and an increase in IFN-–induced chemokines (interferon-induced protein 10 and monokine induced by interferon-).38 Although three partial responses were observed in the latter study, dose-limiting hepatotoxicity and neutropenia were noted; the authors concluded that the recommended doses for further study were IL-12 500 ng/kg and IFN- 1 MU/m2 (both administered SC).38 These studies clearly indicate that the dose and schedule of cytokine combinations must be chosen carefully to avoid toxicity and achieve the desired synergistic antitumor effects.
There has been recent concern that rhIL-12 may soon become unavailable for clinical use. However, a number of preclinical investigations are underway that may provide alternative methods of delivering IL-12 to patients with cancer, including the use of IL-12–expressing viral vectors,39-41 IL-12 fusion proteins,42,43 and direct delivery of the IL-12 gene into tumors.44,45 In addition, several agents that are capable of inducing the endogenous production of IFN- (and, therefore, could be used to enhance Jak-STAT signaling) are currently being evaluated for their anticancer activity in clinical trials. These include toll-like receptor agonists,46 CpG oligonucleotides,47,48 IL-18,49 and IL-21 (Roda et al, manuscript submitted for publication). This study demonstrates that levels of IFN- signaling intermediates can be modulated by cytokine treatments and that these changes can be efficiently monitored via flow cytometry. These results are potentially applicable to other immunotherapeutic agents and suggest that modulation of immune cell signal transduction pathways could enhance the biologic response to exogenously administered cytokines.
The data presented here demonstrate that the regimen of IL-12 plus IFN- was well tolerated in patients with advanced malignancy and that IL-12 pretreatments can effectively modulate Jak-STAT signal transduction in patient PBMCs. The efficacy of this treatment regimen is currently being evaluatedin a phase II trial of patients with advanced malignant melanoma (Cancer and Leukemia Group B 50001).
Authors’ Disclosures of Potential Conflicts of Interest
The authors indicated no potential conflicts of interest.
NOTES
Supported by National Institutes of Health Grant Nos. CA84402, P30-CA16058, 2-UO1 CA-076576-06, K08 CA93518 (C.F.E.), and K24 CA93670 (W.E.C.), The Valvano Foundation for Cancer Research Award, and The Ohio State University Department of Surgery Clinical Science Seed Grant. G.B.L. is a National Research Service Award T32 fellow (5 T32 CA90223-02).
C.F.E. and G.B.L. contributed equally to this work.
Authors’ disclosures of potential conflicts of interest are found at the end of this article.
REFERENCES
Lotze MT, Dallal RM, Kirkwood JM, et al: Cutaneous melanoma, in De Vita V, Hellman S, Rosenberg S (eds): Principles and Practice of Oncology. Philadelphia, PA, Lippincott, Williams and Wilkins, 2001, pp 1499-1547
Lens MB, Dawes M: Interferon alfa therapy for malignant melanoma: A systematic review of randomized controlled trials. J Clin Oncol 20:1818-1825, 2002
Pyrhonen S, Salminen E, Ruutu M, et al: Prospective randomized trial of interferon alfa-2a plus vinblastine versus vinblastine alone in patients with advanced renal cell cancer. J Clin Oncol 17:2859-2867, 1999
Steineck G, Strander H, Carbin BE, et al: Recombinant leukocyte interferon alpha-2a and medroxyprogesterone in advanced renal cell carcinoma: A randomized trial. Acta Oncol 29:155-162, 1990
Rini BI, Halabi S, Taylor J, et al: Cancer and Leukemia Group B 90206: A randomized phase III trial of interferon-alpha or interferon-alpha plus anti-vascular endothelial growth factor antibody (bevacizumab) in metastatic renal cell carcinoma. Clin Cancer Res 10:2584-2586, 2004
Darnell JE Jr, Kerr IM, Stark GR: Jak-STAT pathways and transcriptional activation in response to IFNs and other extracellular signaling proteins. Science 264:1415-1421, 1994
Levy DE, Kessler DS, Pine R, et al: Interferon-induced nuclear factors that bind a shared promoter element correlate with positive and negative transcriptional control. Genes Dev 2:383-393, 1988
Meraro D, Gleit-Kielmanowicz M, Hauser H, et al: IFN-stimulated gene 15 is synergistically activated through interactions between the myelocyte/lymphocyte-specific transcription factors, PU.1, IFN regulatory factor-8/IFN consensus sequence binding protein, and IFN regulatory factor-4: Characterization of a new subtype of IFN-stimulated response element. J Immunol 168:6224-6231, 2002
Hatina VJ, Kralova J, Jansa P: Identification of an intragenic interferon-stimulated response element sequence of the mouse class I major histocompatibility complex H-2Kb gene. Exp Clin Immunogenet 13:55-60, 1996
Ohmori Y, Hamilton TA: The interferon-stimulated response element and a kappa B site mediate synergistic induction of murine IP-10 gene transcription by IFN-gamma and TNF-alpha. J Immunol 154:5235-5244, 1995
Asadullah K, Sterry W, Trefzer U: Cytokine therapy in dermatology. Exp Dermatol 11:97-106, 2002
Belardelli F, Ferrantini M, Proietti E, et al: Interferon-alpha in tumor immunity and immunotherapy. Cytokine Growth Factor Rev 13:119-134, 2002
Lesinski GB, Anghelina M, Zimmerer J, et al: The antitumor effects of IFN-alpha are abrogated in a STAT1-deficient mouse. J Clin Invest 112:170-180, 2003
Lesinski GB, Valentino D, Hade EM, et al: Expression of STAT1 and STAT2 in malignant melanoma does not correlate with response to interferon-alpha adjuvant therapy. Cancer Immunol Immunother 54:815-825, 2005
Gately MK, Renzetti LM, Magram J, et al: The interleukin-12/interleukin-12-receptor system: Role in normal and pathologic immune responses. Annu Rev Immunol 16:495-521, 1998
Trinchieri G: Interleukin-12: A cytokine produced by antigen-presenting cells with immunoregulatory functions in the generation of T-helper cells type 1 and cytotoxic lymphocytes. Blood 84:4008-4027, 1994
Lesinski GB, Badgwell B, Zimmerer J, et al: IL-12 pretreatments enhance IFN-alpha-induced Janus kinase-STAT signaling and potentiate the antitumor effects of IFN-alpha in a murine model of malignant melanoma. J Immunol 172:7368-7376, 2004
Carson WE, Giri JG, Lindemann MJ, et al: Interleukin (IL) 15 is a novel cytokine that activates human natural killer cells via components of the IL-2 receptor. J Exp Med 180:1395-1403, 1994
Parihar R, Nadella P, Lewis A, et al: A phase I study of interleukin 12 with trastuzumab in patients with human epidermal growth factor receptor-2-overexpressing malignancies: Analysis of sustained interferon gamma production in a subset of patients. Clin Cancer Res 10:5027-5037, 2004
Fleisher TA, Dorman SE, Anderson JA, et al: Detection of intracellular phosphorylated STAT-1 by flow cytometry. Clin Immunol 90:425-430, 1999
Lesinski GB, Kondadasula SV, Crespin T, et al: Multiparametric flow cytometric analysis of inter-patient variation in STAT1 phosphorylation following interferon alfa immunotherapy. J Natl Cancer Inst 96:1331-1342, 2004
Levy DE, Lew DJ, Decker T, et al: Synergistic interaction between interferon-alpha and interferon-gamma through induced synthesis of one subunit of the transcription factor ISGF3. EMBO J 9:1105-1111, 1990
Schindler C, Shuai K, Prezioso VR, et al: Interferon-dependent tyrosine phosphorylation of a latent cytoplasmic transcription factor. Science 257:809-813, 1992
Carson WE: Interferon-alpha-induced activation of signal transducer and activator of transcription proteins in malignant melanoma. Clin Cancer Res 4:2219-2228, 1998
Ernstoff MS, Nair S, Bahnson RR, et al: A phase IA trial of sequential administration recombinant DNA-produced interferons: Combination recombinant interferon gamma and recombinant interferon alfa in patients with metastatic renal cell carcinoma. J Clin Oncol 8:1637-1649, 1990
Kurzrock R, Rosenblum MG, Quesada JR, et al: Phase I study of a combination of recombinant interferon-alpha and recombinant interferon-gamma in cancer patients. J Clin Oncol 4:1677-1683, 1986
Opalka B, Wandl UB, Becher R, et al: Minimal residual disease in patients with chronic myelogenous leukemia undergoing long-term treatment with recombinant interferon alpha-2b alone or in combination with interferon gamma. Blood 78:2188-2193, 1991
Wandl UB, Kloke O, Nagel-Hiemke M, et al: Combination therapy with interferon alpha-2b plus low-dose interferon gamma in pretreated patients with Ph-positive chronic myelogenous leukaemia. Br J Haematol 81:516-519, 1992
Osanto S, Jansen R, Naipal AM, et al: In vivo effects of combination treatment with recombinant interferon-gamma and -alpha in metastatic melanoma. Int J Cancer 43:1001-1006, 1989
Creagan ET, Ahmann DL, Frytak S, et al: Recombinant leukocyte A interferon (rIFN-alpha A) in the treatment of disseminated malignant melanoma: Analysis of complete and long-term responding patients. Cancer 58:2576-2578, 1986
Physicians’ Desk Reference. Oradell, NJ, Medical Economics, 2002, pp 3120-3128
Vadhan-Raj S, Al-Katib A, Bhalla R, et al: Phase I trial of recombinant interferon gamma in cancer patients. J Clin Oncol 4:137-146, 1986
Calabrese EJ, Baldwin LA: U-shaped dose-responses in biology, toxicology, and public health. Annu Rev Public Health 22:15-33, 2001
Wigginton JM, Wiltrout RH: IL-12/IL-2 combination cytokine therapy for solid tumours: Translation from bench to bedside. Expert Opin Biol Ther 2:513-524, 2002
Wigginton JM, Komschlies KL, Green JE, et al: Evaluation of the antitumor activity of the interleukin-12/pulse interleukin-2 combination. Ann N Y Acad Sci 795:434-439, 1996
Gollob JA, Veenstra KG, Parker RA, et al: Phase I trial of concurrent twice-weekly recombinant human interleukin-12 plus low-dose IL-2 in patients with melanoma or renal cell carcinoma. J Clin Oncol 21:2564-2573, 2003
Carson WE, Dierksheide JE, Jabbour S, et al: Coadministration of interleukin-18 and interleukin-12 induces a fatal inflammatory response in mice: Critical role of natural killer cell interferon-gamma production and STAT-mediated signal transduction. Blood 96:1465-1473, 2000
Alatrash G, Hutson TE, Molto L, et al: Clinical and immunologic effects of subcutaneously administered interleukin-12 and interferon alfa-2b: Phase I trial of patients with metastatic renal cell carcinoma or malignant melanoma. J Clin Oncol 22:2891-2900, 2004
Triozzi PL, Allen KO, Carlisle RR, et al: Phase I study of the intratumoral administration of recombinant canarypox viruses expressing B7.1 and interleukin 12 in patients with metastatic melanoma. Clin Cancer Res 11:4168-4175, 2005
Narvaiza I, Mazzolini G, Barajas M, et al: Intratumoral coinjection of two adenoviruses, one encoding the chemokine IFN-gamma-inducible protein-10 and another encoding IL-12, results in marked antitumoral synergy. J Immunol 164:3112-3122, 2000
Melero I, Mazzolini G, Narvaiza I, et al: IL-12 gene therapy for cancer: In synergy with other immunotherapies. Trends Immunol 22:113-115, 2001
Li J, Hu P, Khawli LA, et al: ChTNT-3/hu IL-12 fusion protein for the immunotherapy of experimental solid tumors. Hybrid Hybridomics 23:1-10, 2004
Peng LS, Penichet ML, Morrison SL: A single-chain IL-12 IgG3 antibody fusion protein retains antibody specificity and IL-12 bioactivity and demonstrates antitumor activity. J Immunol 163:250-258, 1999
Hanna E, Zhang X, Woodlis J, et al: Intramuscular electroporation delivery of IL-12 gene for treatment of squamous cell carcinoma located at distant site. Cancer Gene Ther 8:151-157, 2001
Harada N, Shimada M, Okano S, et al: IL-12 gene therapy is an effective therapeutic strategy for hepatocellular carcinoma in immunosuppressed mice. J Immunol 173:6635-6644, 2004
Okamoto M, Sato M: Toll-like receptor signaling in anti-cancer immunity. J Med Invest 50:9-24, 2003
Stern BV, Boehm BO, Tary-Lehmann M: Vaccination with tumor peptide in CpG adjuvant protects via IFN-gamma-dependent CD4 cell immunity. J Immunol 168:6099-6105, 2002
Roda JM, Parihar R, Carson WE III: CpG-containing oligodeoxynucleotides act through TLR9 to enhance the NK cell cytokine response to antibody-coated tumor cells. J Immunol 175:1619-1627, 2005
Son YI, Dallal RM, Mailliard RB, et al: Interleukin-18 (IL-18) synergizes with IL-2 to enhance cytotoxicity, interferon-gamma production, and expansion of natural killer cells. Cancer Res 61:884-888, 2001(Charles F. Eisenbeis, Gre)
Yale University, New Haven, CT
ABSTRACT
PURPOSE: To evaluate the safety of sequentially administered recombinant (r) human (h) interleukin-12 (IL-12) and interferon alfa-2b (IFN--2b) in patients with advanced cancer and to determine the effects of endogenously produced IFN- on Janus kinase-signal transducer and activator of transcription (Jak-STAT) signal transduction in patient peripheral-blood mononuclear cells (PBMCs).
PATIENTS AND METHODS: Forty-nine patients with metastatic cancer received rhIL-12 on day 1 and IFN--2b on days 2 to 6 of either a 14-day (n = 43) or a 7-day treatment cycle (n = 6). rhIL-12 was initially administered subcutaneously at a dose of 100 ng/kg, whereas IFN--2b was escalated from 1 to 10 million units (MU) in cohorts of three patients (1, 3, 5, 7, or 10 MU). rhIL-12 was subsequently administered intravenously (IV) in escalating doses (100 to 500 ng/kg) to achieve greater IFN- production. Peripheral blood was drawn for measurement of plasma IFN- and the induction of Jak-STAT signal transduction in PBMCs.
RESULTS: No IL-12–or IFN-–related dose-limiting toxicities were observed. There were no responses in 41 assessable patients. Five patients exhibited stable disease lasting 6 months or longer while on therapy. Optimal induction of IFN- by IL-12 occurred after an IV dose of 250 ng/kg. Patient PBMCs exhibited increased levels of STAT1 after IL-12 administration. The peak level of IFN- achieved with IL-12 therapy correlated with the peak level of intracellular STAT1 in patient PBMCs (r = 0.38, P = .021).
CONCLUSION: The combination of rhIL-12 and IFN--2b can be administered sequentially with minimal toxicity. IV administration of rhIL-12 modulates IFN-–induced Jak-STAT signal transduction in patient PBMCs.
INTRODUCTION
Because renal cell carcinomas (RCCs) are relatively chemotherapy-resistant tumors,1 treatment strategies for metastatic RCC and malignant melanomas have focused on immune-based therapies. Interferon alfa (IFN-) produces overall response rates of 15% to 20% in advanced RCC and melanoma. Also, it is the only agent approved for use as adjuvant therapy after surgical resection of high-risk melanoma.2-4 Recently, IFN--2b was chosen to be the control arm in a new, national, phase III trial of the antiangiogenic agent bevacizumab in patients with advanced RCC.5 Thus, IFN- remains an important therapeutic option in several disease settings, and strategies to improve its activity are warranted.
IFN- regulates gene expression in immune effector cells and tumor cells via activation of the Janus kinase–signal transducer and activator of transcription (Jak-STAT) pathway of signal transduction. The binding of IFN- to its receptor activates two receptor-associated Janus family kinases, Jak1 and Tyk2, which subsequently phosphorylate cytoplasmic transcription factors belonging to the STAT family of proteins (STAT1 and STAT2). These ultimately complex with other DNA binding proteins to initiate the transcription of IFN-–responsive genes within immune cells.6-10 In addition, IFN- can exert direct antiproliferative, antiangiogenic, and proapoptotic effects on tumor cells.11,12 Recent studies by our group have shown that the stimulatory effects of IFN- on host immune cells are critically important to its antitumor actions.13,14
Interleukin-12 (IL-12) is produced primarily by monocytes and macrophages15 and stimulates the proliferation and lytic activity of natural killer cells and cytotoxic T cells. In addition, IL-12 induces the secretion of IFN- by both T cells and natural killer cells and promotes the maturation and activation of type 1 helper T cells, thus favoring the development of cellular immunity and immunologic memory over humoral immunity.16 We have previously shown that IFN- produced in response to IL-12 can upregulate the level of Jak-STAT signaling intermediates in both tumor and immune effector cells. Importantly, the upregulation of critical signaling intermediates sensitized host immune cells to lower doses of IFN-, leading to significantly enhanced survival of tumor-bearing mice.17
We hypothesized that recombinant human IL-12 (rhIL-12) pretreatments would sensitize patients to the antitumor effects of low-dose IFN-, leading to increased efficacy and decreased toxicity. We speculated that IL-12 administration might lead to greater endogenous production of IFN- than what could be achieved with a simple injection of recombinant IFN- alone. Therefore, we conducted a phase I trial of IL-12 followed by IFN- in patients with advanced malignancy. The objectives of this trial were to determine the safety and tolerability of this combination cytokine regimen and to determine the effects of IL-12 pretreatment on IFN- signal transduction pathways using flow cytometric assays. The results of this study suggest that modulation of cytokine signal transduction pathways is feasible in the setting of immune-based cancer therapies.
PATIENTS AND METHODS
Objectives and Eligibility
This National Cancer Institute–sponsored phase I trial was conducted at The Ohio State University Comprehensive Cancer Center from August 1998 to January 2001 under Institutional Review Board approval. The primary objective was to determine the maximum-tolerated dose (MTD) of IFN--2b (INTRON A; Schering Corp, Kenilworth, NJ) when preceded by a single dose of IL-12 in patients with advanced malignancy. The secondary objectives were to measure plasma levels of IFN- and to characterize Jak-STAT signal transduction in patient peripheral-blood mononuclear cells (PBMCs) over the course of therapy. Forty-nine patients between the ages of 23 and 84 years were enrolled onto this study. All patients provided informed consent before treatment. This trial was open to patients with advanced cancer of any histology who had a life expectancy of at least 12 weeks. Prior therapy with IFN- was permitted. Patients were required to have a Karnofsky performance status of 70% and adequate hematopoietic, renal, and hepatic function.
Treatment Protocol
Concerns that continuous administration of IL-12 and IFN--2b might be excessively toxic led to the adoption of a biweekly regimen. rhIL-12 was administered on day 1 of each 14-day treatment cycle, followed by subcutaneous (SC) injections of IFN--2b on days 2 through 6 (Fig 1). Injections of rhIL-12 were initially administered SC at a dose of 100 ng/kg (Table 1). After it was determined that SC doses of IL-12 did not induce significant levels of circulating IFN-, the study was amended to permit the IL-12 to be administered as an intravenous (IV) bolus injection (100, 250, or 500 ng/kg). For each dose of IL-12, the IFN--2b component was dose escalated within cohorts of three patients (1, 3, 5, 7, or 10 million units [MU]/d). Individual patients received the same dose of IL-12 and IFN--2b for the duration of the study. All patients received 12 weeks (six cycles) of therapy unless toxicity or disease progression was observed. Disease response evaluation was performed after every 6 cycles, and patients exhibiting a clinical response or stable disease (SD) were offered the option of continuing therapy until disease progression. Late in the study, the protocol was amended to permit patients to receive cytokine therapy on a 7-day cycle. IL-12 was administered IV at 300 ng/kg to all patients in this cohort.
Management of Toxicity
Toxicity was assessed according to the revised National Cancer Institute Common Toxicity Criteria version 2.0. Dose-limiting toxicity (DLT) was defined as any clearly drug-related grade 3 nonhematologic toxicity that did not resolve after a 2-week rest period or that recurred in subsequent treatment cycles or any clearly drug-related grade 4 nonhematologic toxicity. In the absence of DLT, successive cohorts of three patients were entered onto the protocol at increasing doses of IFN--2b. The MTD was defined as one dose level below the dose at which two or more of six patients experienced DLTs.
Procurement of Peripheral Blood
Approximately 8 to 10 mL of peripheral blood was drawn before each injection of cytokine. PBMCs were separated by density gradient centrifugation with Ficoll-Paque (Amersham Pharmacia Biotech, Uppsala, Sweden), and plasma was snap frozen per protocol.18
Quantitation of Plasma IFN-
Patient plasma samples were analyzed in triplicate for IFN- by enzyme-linked immunosorbent assay using commercially available monoclonal antibody (Ab) pairs as previously described.19 Standard curves were prepared for each plate, and, when possible, all plasma samples from a single patient were run on in parallel to minimize interassay variability.
Intracellular Flow Cytometry for STAT1 Levels
Cryopreserved PBMCs were assayed for total STAT1 using a flow cytometric assay as previously described.20,21 Fixed and permeabilized cells were stained with a murine anti-STAT1 Ab (BD Transduction Laboratories, San Diego, CA) followed by secondary Ab (fluorescein isothiocyanate–conjugated goat antimouse; BioSource International, Camarillo, CA) and then analyzed on a Coulter Elite II flow cytometer (Beckman Coulter, Fullerton, CA).
Statistical Analysis
Data were examined for normal distribution. Fisher’s exact test was used to test for differences in IFN- levels after treatment with high- or low-dose IL-12. Student’s t test was used to test whether STAT1 levels were related to IL-12 dose. A nonparametric Spearman rank correlation was used to test whether IFN- levels and intracellular STAT1 were correlated. P < .05 was used to define statistical significance.
RESULTS
Dose Escalation
The characteristics of all patients enrolled onto this study are listed in Table 1. The first 16 patients received an IL-12 dose of 100 ng/kg SC with minimal toxicity; however, this dose did not routinely induce significant production of IFN- (Fig 2). Therefore, subsequent patients (n = 33) received IV doses of IL-12 starting at 100 ng/kg. IL-12 at a dose of 100 ng/kg IV (n = 4) was an ineffective stimulus for IFN- production, and therefore, the dose was increased to 250 ng/kg (n = 17). The IFN--2b component of therapy was dose escalated to 10 MU/d with no DLT. The trial was then amended to permit administration of IL-12 and IFN--2b on a weekly basis. Six patients (patients 38 to 43) received weekly IL-12 at a dose of 300 ng/kg IV followed by IFN--2b SC on days 2 to 6 of each 7-day cycle at a dose of 1 or 3 MU/d (Table 1 and Fig 1). All patients receiving treatments on a 7-day cycle complained of persistent fatigue and/or weakness (grade 1 or 2) by week 4 of therapy. Although these symptoms were manageable, no objective responses were observed, and the trial was amended to return to a 14-day treatment cycle, with the IL-12 dose set at 500 ng/kg IV (n = 6). The MTD was not reached, and the optimal biologic dose of IL-12 (the dose of IL-12 that induced maximal IFN- levels) was determined to be 250 ng/kg IV (see Systemic Levels of IFN-). At this dose, patients routinely tolerated 1 to 10 MU of IFN--2b without difficulty. For all patients, the median number of cycles received was five (range, one to 18 cycles). Forty-five patients received at least one cycle of therapy and were assessable for toxicity, whereas 41 patients completed two cycles of therapy and, therefore, were assessable for response.
Toxicity
No DLTs were encountered during the course of this study. The regimen was well tolerated, and severe adverse events were uncommon. Table 2 reflects the incidence of grade 3 and 4 toxicities across all treatment cycles. There were no deaths on study. Four patients experienced reversible grade 4 nonhematologic toxicities that were deemed unrelated to the study therapy. Patient 30 developed grade 4 dyspnea during the third cycle of therapy (IL-12 250 ng/kg; IFN--2b 5 MU) as a result of bilateral malignant pleural effusions and was taken off study because of disease progression. Patient 32 developed grade 4 myalgia on day 6 of the first cycle of therapy (IL-12 250 ng/kg; IFN--2b 7 MU) that rapidly resolved. The patient subsequently received a total of nine cycles of therapy without recurrence of the myalgia. Patient 37 (IL-12 250 ng/kg; IFN--2b 10 MU) was receiving hydrochlorothiazide for pre-existing lower-extremity edema and developed asymptomatic grade 4 hyponatremia on day 3 of cycle 1 (118 mg/dL). The serum sodium returned to baseline after discontinuation of hydrochlorothiazide. Patient 40 received six cycles of therapy (weekly regimen) without significant toxicity (IL-12 300 mg/kg; IFN--2b 1 MU). However, during cycle 7, this patient developed Pseudomonas pneumonia and sepsis associated with grade 4 hypoxia and hypotension requiring ventilatory support. This patient was removed from the study. In addition, reversible grade 4 neutropenia occurred in patient 31 (IL-12 250 ng/kg; IFN--2b 7 MU) and patient 41 (IL-12 300 mg/kg; IFN--2b 3 MU, weekly cycle), but it was readily reversible and did not prevent these patients from receiving further cycles of therapy. Less severe toxicities that were commonly encountered included fever, chills, and transient fatigue. These toxicities routinely occurred within 2 to 18 hours of IL-12 administration and were most severe after the first dose of IL-12. Pretreatment with acetaminophen (650 mg) or celecoxib (100 to 200 mg) resulted in fewer and less severe reactions and was routinely used in the latter portion of the trial. The most frequent hematologic toxicities were transient lymphopenia and neutropenia, which occurred more often in patients receiving high doses of IFN--2b ( 7 MU).
Efficacy
Forty-one patients completed two cycles of therapy and were assessable for disease response. No objective responses were seen. Five patients completed at least 12 14-day cycles of therapy (ie, 6 months) and were classified as having SD. Patients 38 and 41 received 12 cycles of therapy on a 7-day cycle (3 months total therapy) and were not considered to have SD. All of the SD patients received IV IL-12, and four of the five patients were treated with IL-12 doses of at least 250 ng/kg (Table 3). However, SD was not statistically associated with plasma levels of IFN- (see next section).
Systemic Levels of IFN-
Plasma IFN- levels were evaluated in all patients before each injection of IL-12 or IFN- (n = 49). In the majority of assessable patients (45 of 49 patients; 91%), peak levels of IFN- occurred on day 2 of the treatment cycle (ie, approximately 24 hours after IL-12 administration) and remained elevated over the next few days. Peak IFN- levels were significantly higher in patients receiving IL-12 at 250 or 300 ng/kg IV compared with patients receiving IL-12 100 ng/kg SC (P < .001, Fig 2). An increase in the IL-12 dose to 500 ng/kg did not lead to further enhancement of IFN- production. Also, 95% of patients (21 of 22 patients) receiving the 250 ng/kg or 300 ng/kg doses of IL-12 had elevated levels of IFN- during every treatment cycle (median number of cycles completed = six) compared with just 37% of patients (six of 16 patients; median cycles completed = 5.5) receiving IL-12 at 100 ng/kg SC (P < .05).
Intracellular Levels of STAT1
We used a flow cytometric assay to test whether endogenous production of IFN- after IL-12 administration led to increased expression of Jak-STAT signaling intermediates in PBMCs, as had been observed in a murine model of melanoma.17 STAT1 levels were measured in PBMCs that were procured on days 1 to 6 of cycle 1. Sufficient data for analysis was available for 23 patients. The remaining 26 patients received their SC injections of IFN- at home, and thus, their PBMCs were not available for analysis. Flow cytometric data from two representative patients are shown in Figures 3A and 3B. Further analysis of STAT1 levels (independent of IL-12 dosage) revealed that the levels of STAT1 were markedly upregulated 1 day after IL-12 administration (day 2 value) and continued to increase until reaching a peak on day 4 (P < .01; analysis of variance). Although STAT1 levels began to gradually decline at this point, they typically remained elevated above baseline on days 5 and 6 of each cycle. A post hoc comparison of this data also demonstrated that day 4 STAT1 levels were significantly higher than the levels measured on days 1 or 2 but not significantly different from STAT1 levels observed on days 3, 5, and 6. This profile of increased STAT1 levels after IL-12 administration was evident for all 23 patients. The trend of increasing STAT1 levels from baseline (day 1) to day 6 is illustrated for patients at each dose level of IL-12 (Figs 3C to 3G).
A plot of the maximal increase in STAT1 over baseline for each dose level of IL-12 (cycle 1 values) is presented in Figure 4A. A comparison of the 100 ng/kg SC and 250 and 300 ng/kg IV dose levels revealed no direct relationship between dosage of IL-12 and total STAT1 protein levels (P = .19). Given our hypothesis that the increase in STAT1 within patient PBMCs was mediated by circulating levels of IFN-, the relationship between these two patient characteristics was examined. A plot of maximum STAT1 increase versus maximum plasma IFN- level in week 1 revealed that these two variables were statistically correlated (r = 0.38, P = .021). This finding indicates that endogenously produced IFN- is likely responsible for modulating levels of STAT1 within patient PBMCs (Fig 4B). However, the observed modifications in the Jak-STAT signal transduction pathway were not clinically beneficial.
DISCUSSION
We have demonstrated that administration of IL-12 followed by IFN- was well tolerated in patients with advanced malignancy. Although no responses were observed in 41 assessable patients, five patients had prolonged stabilization of their disease ( 6 months) during the trial. Correlative studies indicated that maximum IFN- levels were achieved on day 2 of each cycle and that IV administration of IL-12 (250 or 300 ng/kg) induced the maximum level of IFN-. Plasma levels of IFN- correlated with the induction of intracellular STAT1 protein within patient PBMCs during cycle 1. These correlative data are the first to demonstrate that IFN-–induced Jak-STAT signal transduction can be modulated in vivo via IL-12 administration. Furthermore, we have demonstrated the utility of a flow cytometric assay for the analysis of signaling in patient cells after cytokine therapy.
We hypothesized that enhanced expression of Jak-STAT signaling components would render patient immune cells more sensitive to subsequent doses of IFN-. Studies of the molecular mechanism of IFN- pretreatment have indicated that its actions are dose dependent and require protein synthesis, which is a process that may require anywhere from 6 to 24 hours.22-24 Therefore, the optimal schema for the administration of IFN- and IFN- would most likely involve pretreatment of patients with IFN- several hours before the administration of IFN-. This schedule has only been studied in a trial conducted by Ernstoff et al,25 who examined the sequential administration of IFN- and IFN- in patents with advanced RCC. They noted complete responses in two patients (n = 36) and partial responses in an additional six patients. Other phase I studies have shown that coadministration of IFN- and IFN- was well tolerated.25-30
IL-12 was chosen as a pretreatment in this study on the basis of its ability to stimulate the sustained endogenous production of IFN-, which is a factor that can markedly enhance STAT1 expression within immune effector cells and tumor cells.6,17,24 Overall, IL-12 administration was an effective stimulus for IFN- production. We hypothesized that the administration of IL-12 would result in greater endogenous production of IFN- than what would typically be achieved via IV or SC injections of recombinant IFN-. In this trial, IFN- levels remained elevated above baseline for at least 48 hours after a single injection of IL-12. The mean elimination half-life of IFN- is reported to be 38 minutes when administered IV and 5.9 hours when administered as an SC injection. Peak plasma concentrations of IFN- occur at 7 hours after SC dosing.31 In a phase I study of recombinant IFN- in cancer patients, Vadhan-Raj et al32 demonstrated that serum IFN- levels returned to baseline within 2 hours after a 6-hour infusion of IFN- 1 mg/m2. Thus, a single injection of IL-12 leads to prolonged elevations in plasma IFN- that are superior to the elevations that can be achieved via administration of the recombinant cytokine.
The induction of IFN- by IL-12 seemed to be variable within each cohort of patients. The IV route of administration was superior to the SC route for the induction of IFN-, but an increase in the IL-12 dose from 300 to 500 ng/kg did not lead to increased production of IFN-. Although limited patient numbers in some of the dose levels prevented comparisons across treatment groups, this observation suggests that the MTD of IL-12 is not necessarily the most biologically active dose. The apparent reduced efficacy of IL-12 at the highest dose tested may be indicative of hormesis, a U-shaped dose-response phenomenon that is characterized by stimulation at low doses and inhibition at higher doses.33
We have previously demonstrated an essential role for STAT1 signal transduction within host immune cells for mediating the antitumor effects of IFN-.13 Therefore, in this trial, it was of particular interest to determine whether STAT1 levels could be modulated in patients with advanced cancer. Levels of intracellular STAT1 protein in patient PBMCs increased dramatically by day 2 of the cycle, reached a peak on day 4, and then declined over the next few days. Importantly, circulating levels of IFN- correlated with the induction of STAT1 protein within patient PBMCs, suggesting that this cytokine was indeed responsible for the changes that had occurred. This effect of IFN- on the Jak-STAT pathway was consistent with what had been previously reported in the context of in vitro studies conducted by our laboratory.17 This effect may partly explain the synergy that is observed when IL-12 is combined with other cytokines34-37 and may represent a functional pathway for optimization of Jak-STAT signal transduction during infectious processes.
IL-12 has been infrequently administered with other cytokines in clinical trials largely because of the fear of synergistic and/or overlapping toxicities. SC administration of low-dose IL-2 enhanced the IFN- response to IL-12 and prevented the IFN- response from declining over time,36 and IL-12 administered twice weekly with IFN- (thrice weekly) resulted in elevated levels of IFN- and an increase in IFN-–induced chemokines (interferon-induced protein 10 and monokine induced by interferon-).38 Although three partial responses were observed in the latter study, dose-limiting hepatotoxicity and neutropenia were noted; the authors concluded that the recommended doses for further study were IL-12 500 ng/kg and IFN- 1 MU/m2 (both administered SC).38 These studies clearly indicate that the dose and schedule of cytokine combinations must be chosen carefully to avoid toxicity and achieve the desired synergistic antitumor effects.
There has been recent concern that rhIL-12 may soon become unavailable for clinical use. However, a number of preclinical investigations are underway that may provide alternative methods of delivering IL-12 to patients with cancer, including the use of IL-12–expressing viral vectors,39-41 IL-12 fusion proteins,42,43 and direct delivery of the IL-12 gene into tumors.44,45 In addition, several agents that are capable of inducing the endogenous production of IFN- (and, therefore, could be used to enhance Jak-STAT signaling) are currently being evaluated for their anticancer activity in clinical trials. These include toll-like receptor agonists,46 CpG oligonucleotides,47,48 IL-18,49 and IL-21 (Roda et al, manuscript submitted for publication). This study demonstrates that levels of IFN- signaling intermediates can be modulated by cytokine treatments and that these changes can be efficiently monitored via flow cytometry. These results are potentially applicable to other immunotherapeutic agents and suggest that modulation of immune cell signal transduction pathways could enhance the biologic response to exogenously administered cytokines.
The data presented here demonstrate that the regimen of IL-12 plus IFN- was well tolerated in patients with advanced malignancy and that IL-12 pretreatments can effectively modulate Jak-STAT signal transduction in patient PBMCs. The efficacy of this treatment regimen is currently being evaluatedin a phase II trial of patients with advanced malignant melanoma (Cancer and Leukemia Group B 50001).
Authors’ Disclosures of Potential Conflicts of Interest
The authors indicated no potential conflicts of interest.
NOTES
Supported by National Institutes of Health Grant Nos. CA84402, P30-CA16058, 2-UO1 CA-076576-06, K08 CA93518 (C.F.E.), and K24 CA93670 (W.E.C.), The Valvano Foundation for Cancer Research Award, and The Ohio State University Department of Surgery Clinical Science Seed Grant. G.B.L. is a National Research Service Award T32 fellow (5 T32 CA90223-02).
C.F.E. and G.B.L. contributed equally to this work.
Authors’ disclosures of potential conflicts of interest are found at the end of this article.
REFERENCES
Lotze MT, Dallal RM, Kirkwood JM, et al: Cutaneous melanoma, in De Vita V, Hellman S, Rosenberg S (eds): Principles and Practice of Oncology. Philadelphia, PA, Lippincott, Williams and Wilkins, 2001, pp 1499-1547
Lens MB, Dawes M: Interferon alfa therapy for malignant melanoma: A systematic review of randomized controlled trials. J Clin Oncol 20:1818-1825, 2002
Pyrhonen S, Salminen E, Ruutu M, et al: Prospective randomized trial of interferon alfa-2a plus vinblastine versus vinblastine alone in patients with advanced renal cell cancer. J Clin Oncol 17:2859-2867, 1999
Steineck G, Strander H, Carbin BE, et al: Recombinant leukocyte interferon alpha-2a and medroxyprogesterone in advanced renal cell carcinoma: A randomized trial. Acta Oncol 29:155-162, 1990
Rini BI, Halabi S, Taylor J, et al: Cancer and Leukemia Group B 90206: A randomized phase III trial of interferon-alpha or interferon-alpha plus anti-vascular endothelial growth factor antibody (bevacizumab) in metastatic renal cell carcinoma. Clin Cancer Res 10:2584-2586, 2004
Darnell JE Jr, Kerr IM, Stark GR: Jak-STAT pathways and transcriptional activation in response to IFNs and other extracellular signaling proteins. Science 264:1415-1421, 1994
Levy DE, Kessler DS, Pine R, et al: Interferon-induced nuclear factors that bind a shared promoter element correlate with positive and negative transcriptional control. Genes Dev 2:383-393, 1988
Meraro D, Gleit-Kielmanowicz M, Hauser H, et al: IFN-stimulated gene 15 is synergistically activated through interactions between the myelocyte/lymphocyte-specific transcription factors, PU.1, IFN regulatory factor-8/IFN consensus sequence binding protein, and IFN regulatory factor-4: Characterization of a new subtype of IFN-stimulated response element. J Immunol 168:6224-6231, 2002
Hatina VJ, Kralova J, Jansa P: Identification of an intragenic interferon-stimulated response element sequence of the mouse class I major histocompatibility complex H-2Kb gene. Exp Clin Immunogenet 13:55-60, 1996
Ohmori Y, Hamilton TA: The interferon-stimulated response element and a kappa B site mediate synergistic induction of murine IP-10 gene transcription by IFN-gamma and TNF-alpha. J Immunol 154:5235-5244, 1995
Asadullah K, Sterry W, Trefzer U: Cytokine therapy in dermatology. Exp Dermatol 11:97-106, 2002
Belardelli F, Ferrantini M, Proietti E, et al: Interferon-alpha in tumor immunity and immunotherapy. Cytokine Growth Factor Rev 13:119-134, 2002
Lesinski GB, Anghelina M, Zimmerer J, et al: The antitumor effects of IFN-alpha are abrogated in a STAT1-deficient mouse. J Clin Invest 112:170-180, 2003
Lesinski GB, Valentino D, Hade EM, et al: Expression of STAT1 and STAT2 in malignant melanoma does not correlate with response to interferon-alpha adjuvant therapy. Cancer Immunol Immunother 54:815-825, 2005
Gately MK, Renzetti LM, Magram J, et al: The interleukin-12/interleukin-12-receptor system: Role in normal and pathologic immune responses. Annu Rev Immunol 16:495-521, 1998
Trinchieri G: Interleukin-12: A cytokine produced by antigen-presenting cells with immunoregulatory functions in the generation of T-helper cells type 1 and cytotoxic lymphocytes. Blood 84:4008-4027, 1994
Lesinski GB, Badgwell B, Zimmerer J, et al: IL-12 pretreatments enhance IFN-alpha-induced Janus kinase-STAT signaling and potentiate the antitumor effects of IFN-alpha in a murine model of malignant melanoma. J Immunol 172:7368-7376, 2004
Carson WE, Giri JG, Lindemann MJ, et al: Interleukin (IL) 15 is a novel cytokine that activates human natural killer cells via components of the IL-2 receptor. J Exp Med 180:1395-1403, 1994
Parihar R, Nadella P, Lewis A, et al: A phase I study of interleukin 12 with trastuzumab in patients with human epidermal growth factor receptor-2-overexpressing malignancies: Analysis of sustained interferon gamma production in a subset of patients. Clin Cancer Res 10:5027-5037, 2004
Fleisher TA, Dorman SE, Anderson JA, et al: Detection of intracellular phosphorylated STAT-1 by flow cytometry. Clin Immunol 90:425-430, 1999
Lesinski GB, Kondadasula SV, Crespin T, et al: Multiparametric flow cytometric analysis of inter-patient variation in STAT1 phosphorylation following interferon alfa immunotherapy. J Natl Cancer Inst 96:1331-1342, 2004
Levy DE, Lew DJ, Decker T, et al: Synergistic interaction between interferon-alpha and interferon-gamma through induced synthesis of one subunit of the transcription factor ISGF3. EMBO J 9:1105-1111, 1990
Schindler C, Shuai K, Prezioso VR, et al: Interferon-dependent tyrosine phosphorylation of a latent cytoplasmic transcription factor. Science 257:809-813, 1992
Carson WE: Interferon-alpha-induced activation of signal transducer and activator of transcription proteins in malignant melanoma. Clin Cancer Res 4:2219-2228, 1998
Ernstoff MS, Nair S, Bahnson RR, et al: A phase IA trial of sequential administration recombinant DNA-produced interferons: Combination recombinant interferon gamma and recombinant interferon alfa in patients with metastatic renal cell carcinoma. J Clin Oncol 8:1637-1649, 1990
Kurzrock R, Rosenblum MG, Quesada JR, et al: Phase I study of a combination of recombinant interferon-alpha and recombinant interferon-gamma in cancer patients. J Clin Oncol 4:1677-1683, 1986
Opalka B, Wandl UB, Becher R, et al: Minimal residual disease in patients with chronic myelogenous leukemia undergoing long-term treatment with recombinant interferon alpha-2b alone or in combination with interferon gamma. Blood 78:2188-2193, 1991
Wandl UB, Kloke O, Nagel-Hiemke M, et al: Combination therapy with interferon alpha-2b plus low-dose interferon gamma in pretreated patients with Ph-positive chronic myelogenous leukaemia. Br J Haematol 81:516-519, 1992
Osanto S, Jansen R, Naipal AM, et al: In vivo effects of combination treatment with recombinant interferon-gamma and -alpha in metastatic melanoma. Int J Cancer 43:1001-1006, 1989
Creagan ET, Ahmann DL, Frytak S, et al: Recombinant leukocyte A interferon (rIFN-alpha A) in the treatment of disseminated malignant melanoma: Analysis of complete and long-term responding patients. Cancer 58:2576-2578, 1986
Physicians’ Desk Reference. Oradell, NJ, Medical Economics, 2002, pp 3120-3128
Vadhan-Raj S, Al-Katib A, Bhalla R, et al: Phase I trial of recombinant interferon gamma in cancer patients. J Clin Oncol 4:137-146, 1986
Calabrese EJ, Baldwin LA: U-shaped dose-responses in biology, toxicology, and public health. Annu Rev Public Health 22:15-33, 2001
Wigginton JM, Wiltrout RH: IL-12/IL-2 combination cytokine therapy for solid tumours: Translation from bench to bedside. Expert Opin Biol Ther 2:513-524, 2002
Wigginton JM, Komschlies KL, Green JE, et al: Evaluation of the antitumor activity of the interleukin-12/pulse interleukin-2 combination. Ann N Y Acad Sci 795:434-439, 1996
Gollob JA, Veenstra KG, Parker RA, et al: Phase I trial of concurrent twice-weekly recombinant human interleukin-12 plus low-dose IL-2 in patients with melanoma or renal cell carcinoma. J Clin Oncol 21:2564-2573, 2003
Carson WE, Dierksheide JE, Jabbour S, et al: Coadministration of interleukin-18 and interleukin-12 induces a fatal inflammatory response in mice: Critical role of natural killer cell interferon-gamma production and STAT-mediated signal transduction. Blood 96:1465-1473, 2000
Alatrash G, Hutson TE, Molto L, et al: Clinical and immunologic effects of subcutaneously administered interleukin-12 and interferon alfa-2b: Phase I trial of patients with metastatic renal cell carcinoma or malignant melanoma. J Clin Oncol 22:2891-2900, 2004
Triozzi PL, Allen KO, Carlisle RR, et al: Phase I study of the intratumoral administration of recombinant canarypox viruses expressing B7.1 and interleukin 12 in patients with metastatic melanoma. Clin Cancer Res 11:4168-4175, 2005
Narvaiza I, Mazzolini G, Barajas M, et al: Intratumoral coinjection of two adenoviruses, one encoding the chemokine IFN-gamma-inducible protein-10 and another encoding IL-12, results in marked antitumoral synergy. J Immunol 164:3112-3122, 2000
Melero I, Mazzolini G, Narvaiza I, et al: IL-12 gene therapy for cancer: In synergy with other immunotherapies. Trends Immunol 22:113-115, 2001
Li J, Hu P, Khawli LA, et al: ChTNT-3/hu IL-12 fusion protein for the immunotherapy of experimental solid tumors. Hybrid Hybridomics 23:1-10, 2004
Peng LS, Penichet ML, Morrison SL: A single-chain IL-12 IgG3 antibody fusion protein retains antibody specificity and IL-12 bioactivity and demonstrates antitumor activity. J Immunol 163:250-258, 1999
Hanna E, Zhang X, Woodlis J, et al: Intramuscular electroporation delivery of IL-12 gene for treatment of squamous cell carcinoma located at distant site. Cancer Gene Ther 8:151-157, 2001
Harada N, Shimada M, Okano S, et al: IL-12 gene therapy is an effective therapeutic strategy for hepatocellular carcinoma in immunosuppressed mice. J Immunol 173:6635-6644, 2004
Okamoto M, Sato M: Toll-like receptor signaling in anti-cancer immunity. J Med Invest 50:9-24, 2003
Stern BV, Boehm BO, Tary-Lehmann M: Vaccination with tumor peptide in CpG adjuvant protects via IFN-gamma-dependent CD4 cell immunity. J Immunol 168:6099-6105, 2002
Roda JM, Parihar R, Carson WE III: CpG-containing oligodeoxynucleotides act through TLR9 to enhance the NK cell cytokine response to antibody-coated tumor cells. J Immunol 175:1619-1627, 2005
Son YI, Dallal RM, Mailliard RB, et al: Interleukin-18 (IL-18) synergizes with IL-2 to enhance cytotoxicity, interferon-gamma production, and expansion of natural killer cells. Cancer Res 61:884-888, 2001(Charles F. Eisenbeis, Gre)