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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

From the Experimental Therapeutics Program, Cleveland Clinic Taussig Cancer Center, and Department of Immunology, Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, OH

Address reprint requests to Ronald M. Bukowski, MD, Experimental Therapeutics Program, Department of Hematology and Medical Oncology, Taussig Cancer Center, The Cleveland Clinic Foundation, 9500 Euclid Ave, Cleveland, OH 44195; e-mail: bukowsr{at}cc.ccf.org

	ABSTRACT

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION Authors' Disclosures of... REFERENCES

 
PURPOSE: Interleukin-12 (IL-12) and interferon alfa-2b (IFN--2b) are pleiotropic cytokines with activity in renal cell carcinoma (RCC) and malignant melanoma (MM) as single agents. Preclinical studies suggest concurrent administration may have synergistic antitumor effects. We conducted a phase I trial of concurrent subcutaneous (SC) administration of IL-12 and IFN--2b in patients with metastatic RCC or MM to determine toxicity, maximum-tolerated dose, preliminary efficacy, and effects on chemokine/cytokine gene expression in peripheral blood mononuclear cells (PBMCs).

PATIENTS AND METHODS: Cohorts of three to six patients were treated with escalating doses of IL-12 (dose I, 100 ng/kg; dose II, 300 ng/kg; dose III, 500 ng/kg; dose IV, 500 ng/kg SC) given twice weekly and IFN--2b (dose I, 1.0 MU/m²; dose II, 1.0 MU/m²; dose III, 1.0 MU/m²; dose IV, 3.0 MU/m² SC) three times weekly in 4-week cycles. Effects on gene expression were assessed by reverse transcriptase polymerase chain reaction.

RESULTS: Twenty-six patients (19 with RCC, seven with MM) were accrued at dose levels I (n = 3), II (n = 3), III (n = 13), and IV (n = 7). Dose-limiting toxicity included grades 3 and 4 hepatotoxicity and neutropenia/leukopenia. Patients received a median of three cycles of treatment. Two patients with RCC and one patient with MM had partial responses. Median survival was 13.8 months. Reverse transcriptase polymerase chain reaction on PBMCs revealed induction of IP-10, Mig, B7.1 (CD80), interleukin-5, and interferon gamma in selected patients.

CONCLUSION: Concurrent SC administration of IL-12 and IFN--2b is possible at the dose levels utilized. Recommended doses for phase II trials are 500 ng/kg IL-12 and 1.0 MU/m² IFN--2b. Consistent induction of IP-10 and Mig, as well as variable induction of B7.1, interleukin-5, and interferon gamma expression was noted in PBMCs.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION Authors' Disclosures of... REFERENCES

 
Renal cell carcinoma (RCC) and malignant melanoma (MM) account for 5% to 10% of all newly diagnosed cancers in the United States.^1–7 The annual incidence of both cancers has increased steadily over the past decade, with many patients having advanced disease at presentation.^1–4,7 Despite recent advances in cancer therapy, the prognosis for this group of patients remains poor, irrespective of the type of therapy administered.^8–10 The role of systemic therapy has been investigated, but overall response rates are low, with a limited survival improvement.

RCC and MM are tumors refractory to standard chemotherapeutic regimens with poor overall objective response rates.^1–4,6,11 A variety of cytokines have been investigated in preclinical murine tumor models and in patients with RCC or MM. Two of these agents, recombinant human interleukin-2 (rHuIL-2) and recombinant human interferon alfa-2b (rHuIFN--2b), have antitumor effects that are reproducible. The use of these agents in advanced RCC and MM have resulted in objective response rates of 15% to 40%, which were durable in some patients.^12–16 Recent studies have identified other cytokines, such as interleukin-12 (IL-12), that may be as effective as rHuIL-2 and rHuIFN--2b^17–19; however, the optimal regimen has not been determined.

rHuIL-12 is a heterodimeric protein composed of two disulfide-linked subunits having molecular masses of 40 kDa and 35 kDa, respectively.^18,19 IL-12 exerts a number of regulatory effects on numerous cell types, particularly on T-lymphocytes and natural killer (NK)-cells, where it enhances NK/lymphokine-activated killer cell lytic activity, facilitates specific cytolytic T-lymphocyte responses, induces interferon gamma (IFN-) secretion by both T- and NK-cells, and supports cell-mediated immune responses by promoting Th₁-type helper T-cell development.^18–20 The induction of IFN- by IL-12 further contributes to IL-12 mediated antitumor activity secondary to inhibition of tumor angiogenesis, as IFN- induces a variety of antiangiogenic chemokines.^21–23 The antitumor activities of rMuIL-12 (murine IL-12) have been demonstrated in a number of murine tumor models, including Renca (renal cell adenocarcinoma), C26 colon carcinoma, B16F10 malignant melanoma, MCA-105 and MCA-207 sarcomas, Lewis lung carcinoma, and MC-38 colon adenocarcinoma.^20,24–26

Numerous clinical trials have been conducted to determine both toxicity and efficacy of subcutaneous (SC) and intravenous (IV) IL-12 in patients with RCC and MM, resulting in variable maximum tolerated doses (MTDs) ranging from 500 ng/kg to 1,250 ng/kg, determined by predosing, initial dosing, and gradual dose escalation.^27–32 The study reported by Motzer et al²⁷ in patients with RCC found an MTD for SC-administered rHuIL-12 of 1.0 µg/kg (weekly administration), and with gradual dose escalation, this was increased to 1.5 µg/kg. Another study reported by Coughlin et al²⁸ noted that the preadministration of a sensitizing dose of rHuIL-12 permitted eight-fold higher daily doses. Reported toxicity has included constitutional symptoms in over 50% of patients, and grade 3/4 toxicities consisting of hepatic toxicity (bilirubin, AST/ALT), leukopenia/neutropenia, and one report of pulmonary toxicity.^27,28,33

Though the exact antitumor mechanism remains unclear, preliminary studies suggest that IL-12 may mediate its effect in part via the induction of IFN- and the IFN--dependent chemokines IP-10 and Mig.³⁴ We have previously reported that tumors from IL-12-treated animals exhibit high levels of the IFN--inducible chemokines IP-10 and Mig, which were shown to mediate chemotaxis of activated T-cells by virtue of their ability to engage the CXCR3 receptor present specifically on activated T-cells.³⁵ It is thus likely that these chemokines may play an important role in attracting the effector lymphocytes observed in IL-12-treated regressing tumors. Tannenbaum et al³⁴ reported that anti-IP-10 and anti-Mig antibodies, when administered to tumor-bearing animals during the course of IL-12 therapy, abrogate T-cell infiltration into the treated tumor, thereby significantly inhibiting perforin and granzyme expression within the tumor bed, and markedly interfering with IL-12 antitumor activity as assessed by tumor growth. These studies support the notion that at least one component of the IL-12 antitumor effect is exerted via the IFN- mediated induction of the chemokines IP-10 and Mig.

rHuIFN--2b has been frequently used in the treatment of RCC and MM. Originally described as an antiviral protein, this pleotropic cytokine has multiple antiproliferative and immunomodulatory biologic effects, in addition to its antiviral activities. Among its numerous immune regulatory mechanisms, IFN- contributes to antitumor immunity by promoting a Th1 immune response,³⁶ upregulating the IL-12 receptor on subsets of lymphocytes,³⁷ and inducing IFN- production by other effector cells.³⁸ This cytokine is tolerable as outpatient therapy and is currently administered SC or by IV. IFN- has been used as a single agent, though it can be combined with a variety of other agents to increase its efficacy in the treatment of both RCC and MM, with varying reports of responses and dose-related toxicities.^{13,16,39–44}

The rationale for the combination of IL-12 and rHuIFN--2b is derived from the prior reports showing the immune-enhancing effects of these cytokines individually, and their limited therapeutic efficacy when used as single agents for cancer therapy, as discussed above. Taken together, previous data suggest that combining rHuIL-12 with rHuIFN--2b may prove to be useful in the treatment of malignant diseases. In this phase I trial, we evaluate the concurrent administration of rHuIL-12 and rHuIFN--2b to determine: (1) the toxicity associated with subcutaneous administration; (2) maximum tolerated doses; (3) effects on chemokine and cytokine gene expression in peripheral blood lymphocytes; and (4) antitumor efficacy.

	PATIENTS AND METHODS

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION Authors' Disclosures of... REFERENCES

 
Patients
Patients in the study had a histologic or cytologic diagnosis of RCC or MM, and strong clinical evidence or biopsy proof of metastases to a site or sites distant from the primary tumor. Patients were required to have bidimensionally measurable or assessable disease; a life expectancy of 3 months; an Eastern Cooperative Oncology Group (ECOG) performance status < 1; recovered from the toxicity of previously administered hormonal, radiation, biologic therapy, or chemotherapy; absence of significant effusions and/or ascites; no major surgery requiring general anesthesia within the past 28 days; and less than three prior treatment regimens. Patients were required to have the following pretreatment laboratory findings above stated minimum values: WBC 3.0 x 10⁹/L, platelets 100 x 10⁹/L, hemoglobin 9.5 gm/100 mL, serum creatinine < 1.8 mg/dL; total bilirubin 1.5 mg/dL; calcium < 11.5 mg/dL; liver enzymes (ALT and AST) < 3x normal, prothrombin time (PT)/partial thromboplastin time (PTT) within institutional normal values. All patients were informed about the investigational nature of this study and written informed consent was obtained in accordance with institutional and federal guidelines.

Exclusion criteria included the following conditions: a history of a serious cardiac arrhythmia, congestive heart failure, angina pectoris, or other severe cardiovascular disease producing limitations of physical activity (ie, New York Heart Association Class III or IV); active peptic ulcer disease, autoimmune disease, or inflammatory bowel disease; local or systemic infections requiring IV antibiotics within the past 28 days; pregnant or lactating women, and fertile women or men unless surgically sterile or using effective contraception; known CNS metastases or known seizure disorder; positive for HIV, hepatitis B antigen, or hepatitis C antigen; history of a malignancy other than a renal cell carcinoma or melanoma (exceptions basal or squamous cell carcinomas of the skin, carcinoma-in-situ of the uterine cervix, and any malignancy treated with curative intent and in complete remission for 3 years); and patients with organ allografts.

All inclusion and exclusion criteria were assessed within 14 days before initiation of therapy, with the exception of x-ray studies not required for determination of tumor measurements, or laboratory studies not used for organ evaluation, which were performed within 28 days of therapy initiation.

Study Drugs
rHuIFN--2b was supplied by the National Cancer Institute (NCI, Bethesda, MD)/Schering (Kenilworth, NJ) as a 5 million U/vial or 10 million U/vial lyophilized powder, stored under refrigeration. The powder was reconstituted with 1 mL of sterile bacteriostatic water for injection before administration. rHuIL-12 was supplied by NCI/Genetics Institute (Cambridge, MA) as a lyophilized drug product containing 50 µg of rHuIL-12 in a 50 mL vial. The lyophilized product was reconstituted with sterile water and administered within 3 hours of initial reconstitution. rHuIL-12 and rHuIFN--2b were administered by standard SC injection.

Dose Schedule
Cohorts of three to six eligible patients were treated with escalating doses of rHuIL-12 (dose I, 100 ng/kg SC, dose II, 300 ng/kg SC, dose III, 500 ng/kg SC, dose IV, 500 ng/kg SC) given twice weekly, and rHuIFN--2b (dose I, 1.0 MU/m² SC, dose II, 1.0 MU/m² SC, dose III, 1.0 MU/m² SC, dose IV, 3.0 MU/m² SC) three times weekly in 4-week cycles until MTD or progression (Table 1). One patient treated at dose level III initially received rHuIL-12 300 ng/kg and rHuIFN--2b 3.0 MU/m² (four doses), with subsequent doses of both cytokines, as outlined. rHuIFN--2b was given 15 minutes after rHuIL-12 at sites including the inferior abdominal wall or extremities, while avoiding sites of previous rHIL-12 administration. rHIL-12 was given SC in the deltoid area of the upper arm; the anteromedial thigh was the alternate site for administration.

Dose-limiting toxicity (DLT) was defined as the occurrence of any of the following during cycle one: (1) grade 3 or higher nonhematologic toxicity; (2) grade 4 neutropenia for 7 days associated with fever or infection; (3) other grade 4 hematologic toxicity. The MTD was defined as the dose level below which two or more of six patients experienced DLT. After DLT was observed in two or more patients at a given dose level, no additional patients were entered at that or higher dose levels.

Gene Expression Studies
Peripheral blood mononuclear cells (PBMCs) from patients were obtained before and at distinct time points (2, 6, and 24 hours, and 7 days) after administration of rHuIL-12 and rHuIFN--2b during cycle one. Isolation of PBMCs and peripheral blood T-cells was performed using density-gradient (Ficoll-Paque^TM PLUS; Amersham Pharmacia Biotech AB, Uppsala, Sweden) centrifugation. Peripheral blood T-cells were purified by negative magnetic selection using microbeads coated with antibodies to CD14, CD16, CD19, CD56, and glycoporin A (Stem Cell Technology, Vancouver, Canada). Isolated cells were processed immediately, and RNA was extracted by the guanidine-isothicyanate/cesium chloride method followed by ethanol precipitation and storage at –70°C until mRNA analysis. Patient selection for these studies was based on patient willingness to participate.

mRNA Analysis of Gene Expression by Reverse Transcriptase Polymerase Chain Reaction (RT-PCR)/Southern Hybridization
Semiquantitative RT-PCR analysis of GADPH, CD80, Mig, and IP-10 mRNA expression in PBMCs was performed using AMV reverse transcriptase (RT) and specific antisense primers (20 µmol/L) at 42°C for one hour followed by polymerase chain reaction (PCR) amplification in a Perkin Elmer/Cetus DNA Thermal Cycler (Perkin-Elmer, Wellesley, MA) for 35 cycles at 94°C for denaturation, 60°C for annealing fragments, and at 72°C for DNA synthesis of the RNA fragments. The following sense and antisense primers were used: (1) GADPH sense: 5'-GAAGGTGAAGGTCGGAGTC-3' and antisense 5'-GAAGATGGTGATGGGATTTC-3'; (2) IP-10 sense: 5'-CCTGCAAGCCAATTTTGTC-3' and antisense 5'-CATTAACCTTCCTACAGGAG-3'; (3) Mig sense: 5'-AGTGGTGTTCTTTTCCTCTTGGG-3' and antisense 5'-CCTTCACATCTGCTGAATCTGGGG-3'; (4) B7.1 (CD80) sense: 5'-ACATGAAGCTGTGGTTGGTTGTC-3' and antisense 5'-GCTGGTCTTTCTCACTTCTGTTCAG-3'. Following amplification and electrophoresis, the gels were blotted overnight onto nitrocellulose membranes and subsequently visualized by Southern hybridization with ³²P-labeled cDNA encoding the desired single-stranded oligonucleotide (GADPH, IP-10, and CD80) or double stranded (Mig) portion of the gene of interest. After overnight hybridization at 42°C, the blots were washed in standard saline-citrate solution and exposed to x-ray film. The probe sequences used were as follows: (1) GADPH: 5'CAAGGTTCCCGTTCTCAGCCTTGACGGTG-3'; (2) IP-10: 5'-TACTAATGCTGATGCAGGTACAGCG-3'; (3) Mig: 5'-GGAAGGGCTTGGGGCAAATTGTTTAAGGTCTTTCAAG-3'; and (4) CD80: 5'-ATAACGTCACTTCAGCCAGGTGTTCCCG-3'.

RNA Isolation and cDNA Synthesis for Real-Time Reactions
Total RNA was isolated from each sample of peripheral blood with silicagel-based membranes (RNeasy; Qiagen, Hilden, Germany) according to manufacturer's protocol. To remove DNA contamination, the samples were incubated with DNase I (0.5 U/10 µg of RNA; Ambion, Austin, TX) at 37°C for 30 minutes. RT was performed using TaqMan RT reagents (Applied Biosystems, Foster City, CA) following a standard protocol, with random hexamers as primers. cDNA synthesis was carried out in a DNA Thermal Cycler (Perkin-Elmer). Patient selection for these studies was based on patient willingness to participate.

Real-Time Quantitative PCR
Using the Primer Express software (Applied Biosystems), primers were designed with amplicons less than 200 bp to enhance efficiency of PCR amplification. Human glucuronidase was identified as the housekeeping gene showing the smallest fluctuation between tested samples, and was thus selected as the internal control.

Amplification was carried out using SYBR Green PCR reagents (Applied Biosystems) according to manufacturer's protocol and 2 µL cDNA. Real-time PCR was performed on an ABI PRISM 7700 Sequence Detector (Perkin-Elmer). The conditions for the PCR amplification were as follows: one cycle at 50°C for 2 minutes, one cycle at 95°C for 10 minutes, 40 cycles at 95°C for 15 seconds, and one cycle at 60°C for 5 minutes. The temperature was then increased from 60°C to 95°C gradually for 20 minutes, and the fluorescence was recorded every 15 seconds to construct the melting curve. All samples were analyzed in triplicate. The following sense and antisense primers were used: (1) huGUS sense 5'-TCATTGGAGGTGCAGCTGAC-3' and antisense 5'-ACTGGCTCTTGGTGACAGCC-3'; (2) IL-5 sense 5'-TCATCGAACTCTGCTGATAGCC and antisense 5'-TTTGACTCTCCAGTGTGCCTATTC-3'; (3) IFN- sense 5'-TTTCAGCTCTGCATCGTTTTG-3' and antisense 5'-TCCGCTACATCTGAATGACCTG-3'. Data shown in Figure 1 and Table 2 represent relative gene expression derived by using the pretreatment (day 0) values as baseline for the measured values obtained on the specified days after treatment.

View larger version (28K): [in this window] [in a new window]   Fig 1. Cytokine expression following treatment. Real-time polymerase chain reaction demonstrating effects of treatment on expression of (A) interleukin-5 and (B) interferon gamma among peripheral blood T cells from patients with renal cell carcinoma (n = 7) and malignant melanoma (n = 1; patient No. 9) following first dose of interleukin-12/interferon alfa-2b. Patients 13 and 14 received dose level IV; others received dose level III.

	RESULTS

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION Authors' Disclosures of... REFERENCES

With the increasing number of cycles administered, an increase in severity of toxicity was noted at dose levels III and IV. No patients at dose levels I or II experienced cumulative toxicity of grade 3 or worse. The most common grade 3 or worse toxicity noted during subsequent cycles was hematologic (neutropenia and/or leukopenia). Treatment-related cumulative toxicities resulted in dose reduction in six patients (n = 2, dose level III; n = 4, dose level IV).

Although the MTD and DLTs are determined by cycle 1 toxicities, it is noteworthy to report one case of severe hepatotoxicity (grade 4 elevations in the transaminases AST, ALT, and GGT, as well as grade 4 elevations in alkaline phosphatase and bilirubin) that was noted in one of the MM patients during cycle 2 at dose level 4. Interestingly, this patient had initially experienced a partial response after the first cycle of treatment. Such elevations in hepatic transaminases have been previously reported following IL-12 therapy for MM.^29,32,33 This hepatotoxicity was completely reversible after withholding therapy. The mechanism of this toxicity remains unclear, as the patient had no clinically apparent liver disease before enrollment. Although subsequent serologic studies revealed past exposure to hepatitis-A and a history of moderate alcohol consumption, neither can adequately explain the observed increases in liver function tests. Trichrome stain of the patient's liver tissue (Fig 2A) reveals extensive fibrosis characteristic of either alcoholic or nonalcoholic steatohepatitis, while immunostaining (Fig 2B) demonstrates macrophage and T-cell infiltration of liver tissue.

View larger version (122K): [in this window] [in a new window]   Fig 2. Liver biopsy demonstrating severe hepatotoxicity. (A) Hematoxylin and eosin stain of liver biopsy containing a part of a cirrhotic nodule; (B) immunohistochemical staining showing increased numbers of T-cells (brown) and macrophages. The pan T-cell marker anti-CD3 accounts for the brown staining in the slide.

Gene Expression
We previously showed that the chemokines IP-10 and Mig can be synthesized by both fresh renal cancer cells and nonseparated PBMCs from IL-12-treated renal cell cancer patients.⁴⁵ In this study, we now show that expression of IP-10 and Mig in PBMCs follows a time course that peaks 6 hours following SC administration of rHuIL-12 and rHuIFN--2b, and declines thereafter (Fig 3). However, though there is an increased expression of IP-10 and Mig following 6 hours of treatment, individual patterns of expression for each patient and chemokine also were seen. IP-10 reached maximal expression after 6 hours and declined or disappeared 7 days after the first dose of rHuIL-12 and rHuIFN--2b administration in all patients (n = 8), whereas Mig chemokine showed sustained expression after 24 hours and through 7 days of treatment in some patients (n = 4), but not in others (n = 4), where declining or weak expression is found after 24 hours or 7 days (Fig 3).

View larger version (102K): [in this window] [in a new window]   Fig 3. Chemokine expression following treatment. Time-course expression of chemokines IP-10 and Mig in unseparated peripheral blood mononuclear cells of a group of patients (n = 8) with advanced renal cell carcinoma following first dose of interleukin-12/interferon alfa-2b immunotherapy. Patients 1 to 3 received dose level I, patients 4 to 6 received dose level II, and patients 7 to 8 received dose level III. hs, hours.

View larger version (79K): [in this window] [in a new window]   Fig 4. Expression of B7.1 (CD80) following treatment. Time-course expression of CD80 among unseparated peripheral blood mononuclear cells of a group of patients (n = 8) with advanced renal cell carcinoma following first dose subcutaneous immunotherapy with interleukin-12/interferon alfa-2b. Patients 1 to 3 received dose level I, patients 4 to 6 received dose level II, and patients 7 to 8 received dose level III. hs, hours.

	DISCUSSION

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION Authors' Disclosures of... REFERENCES

 
The cytokines IL-12 and IFN--2b have been well characterized for their antitumor properties. Phase I trials using IL-12 have shown toxicities consisting mainly of constitutional symptoms (headache, fatigue, myalgias, and arthralgias) in over 50% of patients, as well as grade 3/4 toxicities including hepatic toxicity (bilirubin, AST/ALT), leukopenia/neutropenia, and one report of pulmonary toxicity.^27,29,48 In one trial, the MTD using a fixed dose of IL-12 was 1.0 µg/kg, and with dose titration reached 1.5 µg/kg.²⁷ Similarly, numerous trials have been conducted to investigate the toxicity associated with IFN--2b.^49–51 DLT was predominantly hematologic, including severe neutropenia/leukopenia, as well as gastrointestinal and constitutional symptoms. Toxicities were dose and time dependent, and symptoms subsided within 4 weeks of discontinuation of therapy. It is noteworthy to mention that most trials with IFN--2b have employed doses much higher than the doses used in our study.

The MTD doses noted in this phase I trial for twice weekly SC IL-12 and three times weekly SC IFN--2b were 500 ng/kg and 1 MU/m², respectively. The MTD found for IL-12 was similar to that determined in other studies using IV and SC IL-12;^29–31 however, the MTD reported in this study was two-fold lower than our previously reported MTD for a once weekly administered fixed dose SC IL-12,²⁷ as well as a previously reported MTD for three times weekly SC IL-12.³⁰ The observed differences may be secondary to cumulative toxic effects of both cytokines, as the dose of IL-12 was the same at dose levels III (MTD) and IV, while the IFN--2b at dose level IV was three-fold higher. The addition of IL-12 also contributes to the lower MTD noted for IFN--2b (up to eight-fold lower than in other studies, both as a single agent and in combination with chemotherapeutic agents.^39,40,49,51)

In view of the hepatotoxicity that was noted using the combination of IL-12 and IFN--2b, the coadministration of potentially hepatotoxic drugs at the proposed MTD should ideally be avoided; however, frequent evaluation of liver function tests should be considered if the use of such medications is required. In patients experiencing severe toxicity, the risks and benefits of continuing or withdrawing therapy should be carefully evaluated.

The CXC chemokines Mig and IP-10 have been shown to play an important role in antitumor immunity by acting as chemoattractants for effector cells, including activated CD8+ T-cells. We have previously reported an increase in these chemokines with IL-12 administration as a single agent.⁴⁵ The upregulation of both chemokines was noted in the patient samples studied in this trial (Fig 3). Although we did not look into the additive effects attributable solely to IFN--2b, our results demonstrate that the coadministration of IFN--2b with IL-12 does not abrogate previously reported increases in Mig and IP-10, noted following single-agent IL-12 therapy.⁴⁵ These findings further stress the potential role of cytokine immunotherapy in increasing effector cell trafficking into tumor tissue by inducing chemokine production, which itself inhibits tumor progression, since both Mig and IP-10 are purportedly potent inhibitors of angiogenesis.^52,53 In one patient with a partial response (patient No. 3), there was an early significant increase in both Mig and IP-10 gene expression (Fig 3).

Enhanced immunity with combined IL-12/IFN--2b therapy is further demonstrated by the upregulation of CD80, the T-cell costimulatory glycoprotein found on antigen presenting cells which binds to the CD28 receptors on naive T-cells, which may be an important mediator of IL-12/IFN--2b antitumor immunity (Fig 4). Increased CD80 expression has been associated with increased immunogenicity of tumor cells, while decreased numbers of CD80 expressing cells and/or dysfunctional CD80 expression were noted in some cancers, and were associated with a worse prognosis and an increase in cancer relapse.^54–56 Our studies demonstrate an upregulation of CD80 in some patients (n = 4), which includes one of the patients who was noted to achieve a partial response to therapy (patient No. 3).

In addition to chemokine and costimulatory ligand upregulation, an increase in cytokine production by PBMCs was also noted, specifically IFN- and IL-5 (Figs 1A and B). In contrast to IFN-, which is associated with enhanced antitumor immunity,^57–59 many report that IL-5 has a negative role in tumor immunity by inhibiting cytotoxic T-lymphocyte antitumor responses^60,61; nevertheless, there are a few studies that link IL-5 and Th2 responses to effective tumor immunity.^62,63 IFN- has been shown to promote a Th1 response in part by upregulating IL-12 receptor on Th1 cells, further promoting a Th1 response.³⁷ In this trial, a partial response was noted in the patient who displayed the largest increases in both IL-5 and IFN-; however, it is difficult to determine if the response was due to increases in IL-5, IFN-, or both. The effects of IL-12/IFN--2b on IFN- may also contribute to further tumor infiltration by activated effector cells, based on the induction of the chemokines Mig and IP-10 by IFN-.^64–66 In contrast to previously reported attenuation of IFN- induction by IL-12 after repeated dosing,²⁹ this pattern was not noted in our studies. Furthermore, unlike IL-2, which was recently noted to restore IFN- induction after concurrent administration with IL-12 during later cycles,³² IFN--2b does not appear to alter the pattern of IFN- induction by IL-12.

Both IL-12 and IFN--2b have been reported to independently regulate numerous cytokines, including tumor necrosis factor-, interleukins-2, 4, 6, 8, and 10, as well as various immune cell types.^67–70 The combination of IL-12 and IFN--2b may alter the regulation of cytokines and immune cells that were noted with the use of these two cytokines as single agents. Additional mechanistic studies relating the effects of SC IL-12 and IFN--2b are underway to further elucidate the roles of these cytokines and to investigate their efficacy in patients with MM or RCC.

The present study demonstrates the tolerability of concurrent SC IL-12 and IFN--2b and provides evidence of complimentary immunologic effects warranting further study. The toxicity profile, antitumor activity, and immunologic effects compare favorably to other cytokine combinations utilizing IV IL-12, with the advantage of SC administration. The recommended dose for further study of this combination in patients with RCC and MM is IL-12 at 500 ng/kg and IFN--2b at 1 MU/m².

	Authors' Disclosures of Potential Conflicts of Interest

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION Authors' Disclosures of... REFERENCES

 
The following authors or their immediate family members have indicated a financial interest. No conflict exists for drugs or devices used in a study if they are not being evaluated as part of the investigation. Acted as a consultant within the last 2 years: Ronald M. Bukowski, Schering-Plough. Received more than $2,000 a year from a company for either of the past 2 years: Ronald M. Bukowski, Schering-Plough.

	NOTES

 
Supported by a grant from Schering- Plough and the Zito Chair for Cancer Research.

Authors' disclosures of potential conflicts of interest are found at the end of this article.

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Submitted October 8, 2003; accepted April 28, 2004.

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