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[CANCER RESEARCH 48. 3875-3881. July 1. 1988] A Phase I Study of Recombinant Interleukin 2 plus Recombinant ^-Interferon Robert L. Krigel,1 Kristin A. Padavic-Shaller, Alfred R. Rudolph, Samuel Litwin, Michael Konrad, Edward C. Bradley, and Robert L. (omis Departments of Medicine and Biostatistics, Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111 [R. L. K., K. A. P-S., S. L., K. L. C.], and Cetus Corporation, Emeryville, California 94608 [A. R. R., M. K., E. C. B.J ABSTRACT Interleukin 2 (IL-2) therapies have antitumor activities against several neoplasms. In vitro these activities are enhanced by 0-interferon (IF V tf ). Therefore, we initiated a Phase I trial with a combination of IL-2 and IFN-/3 three times weekly. The IFN-/3 was administered i.v. Initially, the IL-2 was administered s.c. However, neutralizing antibody to the IL-2 developed in five patients, and the route of administration of the IL-2 was changed to i.v. Forty-seven patients were entered on the study. The maximum toler ated doses for the combination given i.v. were 5x10" units/m2 of IL-2 and 10 x IO6 units/m2 of IFN-/3. Dose-limiting toxicities were profound fatigue/decreased performance status, anorexia/weight loss, depression, and arthralgias. Hypotension, exfoliative skin rash, thrombocytopenia, diarrhea, temperature >40.6°C, and peripheral edema were rarely dose limiting. Thirty-two activity to levels higher than those seen with either substance alone (19). Interestingly, IFN-/3 but not a-interferon was syn ergistic with IL-2 in the cytotoxic T-cell mediated destruction of autologous transformed cells (20). In addition, IFN-/3 is also synergistic with 7-interferon, a lymphokine which is generated by cytotoxic T-lymphocytes stimulated by IL-2 (21-23). We now report the results of a Phase I study combining IL2 with IFN-0 which demonstrate the following: (a) that IL-2 and recombinant IFN-ßcan be given together at doses that are tolerable; (b) that IL-2 and recombinant IFN-/3 can generate LAK activity in vivo; and (c) that IFN-7 can be generated with IL-2 with this therapy. MATERIALS patients were évaluablefor response. AND METHODS After 4 weeks of treatment, 21 patients had stable disease, three patients had a minor response, and one patient had a partial response. Significant lymphokineactivated killer cell (LAK) activity was seen in seven patients (22%) and required S x 10* units/m2 of IL-2. Those who had progressive disease had significantly less LAK activity than those with either stable disease or a response. This therapy also induced more than 60 units/ml of endogenous -v-interferon 4 h after the i.v. IL-2 administration. This study demonstrates that (a) intermittent i.v. bolus IL-2 therapy can generate LAK activity, (b) LAK activity may be associated with an antitumor response, (c) significant levels of -y-interferon are induced by this therapy, and (</) IL-2 and IFN-/J given three times weekly i.v. is both tolerable and biologically active. The recommended Phase II dose is 5 x 10' units/m2 of IL-2 plus 6 x 10' units/m2 of IFN-/8. INTRODUCTION Interleukin 2-based treatments have activity against a variety of cancers in both experimental animals and humans (1-6). However, IL-22 therapy has been associated with significant toxicity which has been shown to be directly related to the dose of IL-2 and the duration of its administration. These trials have often included the coadministration of ex vivo IL-2-activated autologous peripheral blood lymphocytes (lymphokine-activated killer cells). It is unclear whether the use of LAK adds to the efficacy of IL-2 used alone and how much LAK contributes to toxicity. Therefore, many investigators are examining alter native dose regimens, schedules, and routes of administration in order to minimize toxicity and improve efficacy without the coadministration of ex vivo activated LAK (5-9). ^-Interferon is a type I interferon, a class of interferons which has shown activity in a wide spectrum of diseases (10-12). IFNßhas also been shown to be synergistic with IL-2 both in stimulating NK activity and in increasing expression of IL-2 receptors (13-18). In patients with AIDS the combination of type I interferons and IL-2 significantly increases the NK Received 11/24/87; revised 3/16/88; accepted 3/31/88. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1To whom requests for reprints should be addressed, at Department of Medicine, Fox Chase Cancer Center, Philadelphia, PA 19111. 3The abbreviations used are: IL-2, human recombinant interleukin 2; II-N. interferon; LAK, lymphokine-activated killer cell(s); NK, natural killer cell; PD, progressive disease; SD, stable disease; ELISA, enzyme-linked immunosorbent assay; LU, lytic units,,, . Patient Population and Treatment Plan The patients selected for this study had histologically proven solid tumor malignancies which were refractory to standard therapy or for which there was no generally accepted therapy. All patients had meas urable or évaluabledisease; Karnofsky performance status >70; ade quate bone marrow (granulocytes, >l,000/mm3, platelets, >50,000/ mm1); renal (creatinine, <2.0 mg/dl) and hepatic (bilirubin, <1.5 mg/ dl) function; and a minimum life expectancy of at least 3 months. Patients with clinically significant cardiac disease, active infection requiring systemic antibiotic therapy, or central nervous system métas tases were excluded from entry. Throughout the duration of the trial, patients did not receive other anticancer treatments, aspirin, nonsteroidal antiinflammatory drugs, corticosteroids, cimetidine, ranitidine, bar biturates, or other investigational agents. Initially, patients were treated with II \ ,< as a 1 min i.v. bolus immediately followed by IL-2 given s.c. During the course of the study, changes were made in the route of administration due to the develop ment of neutralizing antibody in several patients to the s.c. administered IL-2 and after dose-limiting formulation problems which prevented further IL-2 dose escalations. Subsequently, IL-2 was given i.v. as a 1min bolus injection. The first 35 patients received IL-2 s.c. and the last 12 received it i.v. Treatments were administered three times weekly for 4 weeks in the absence of progressive disease or unacceptable toxicity. Patients who exhibited either stable disease or tumor regression were continued on therapy until there was evidence of tumor progression or unacceptable toxicity. The protocol schema is shown in Table 1. Toxicity Criteria The toxicity scale was a modification of the World Health Organi zation criteria. Patients exhibiting Grade IV toxicity were removed from the study. Grade IV toxicity included the following: granulocyte decrease to <250/mm3; platelet decrease to <20,000/mm3; bilirubin, >3.0 mg/dl: serum glutamic and oxaloacetic transaminases, >10x baseline; creatinine. >3.5 mg/dl; intractable vomiting necessitating i.v. fluids; diarrhea requiring i.v. fluids: >10% weight loss; >30-point decrease from baseline Karnofsky performance status; profound motor weakness or autonomie dysfunction; coma or seizures; temperature >40.6°C;or symptomatic fluid accumulation with weight gain >20%. If Grade III toxicity was evident, doses of both agents were reduced by 50%. Grade III toxicity included the following: granulocyte decrease to >250/mm:1 and <500/mm3; platelet decrease to >20,000/mm3 and <40,000/mm3: bilirubin. >2.5 mg/dl but <3.0 mg/dl; serum glutamic and oxaloacetic transaminases, >6 but <10x the baseline; creatinine, >3 mg/dl but <3.5 mg/dl; 6-10 episodes of emesis/day; >7 episodes of diarrhea/day despite antidiarrhea medications; a >20-point decrease 3875 Downloaded from cancerres.aacrjournals.org on June 15, 2017. © 1988 American Association for Cancer Research. PHASE I STUDY OF IL-2 AND IFN-0 Table 1 Protocol schema (s.c.) (units/in2)10"10'10'5X Dose levelIL-2 S.C.IIIIIIIVVVIVIIIL-2 i.v.VVIVIIIL-2 (i.v.) (units/in2)2x 10'5 10'5 x 10'5x x 10'5 10'2x 10'2x 10'2x IO66x 10'10 10'20 x 10'6x x 10«5 x 10'5x x 10'IFN-/3 10'10 10'20 x x 10' from baseline Karnofsky performance status; marked decrease of deep tendon reflexes or marked motor weakness or sensory deficits; confu sion or disorientation; temperatures >39.4°Cbut <40.6°Clasting 3 h or more; weight gain >15% but <20% associated with peripheral edema; ascites or pleural effusion while asymptomatic. If Grade III toxicity continued at the reduced dose, the dose of IFN-/3 was again reduced by 50%. If toxicity continued or progressed to Grade IV, the patient was removed from study. Four patients were entered at each dose level. If any 2 of 4 patients at a dose level experienced Grade III or IV toxicity, an additional 4 patients were entered at that dose level. The maximum tolerated dose was defined as that dose at which 3 of 8 treated patients experienced Grade III or IV toxicity. Response criteria were as follows: partial response required a mini mum of 50% reduction in the sum of the products of the perpendicular diameters of all measurable lesions; minor response was defined as a 25-50% decrease in the sum of the products of the perpendicular diameters of all measurable lesions; stable disease constituted a less than 25% change in tumor measurements; and progressive disease was defined as a 25% or greater increase in the measurable disease. ELISA was used to quantitate levels of antibodies specific to IL-2 or IFN in patient sera (25, 26). Separate assays were done to measure IgG and IgM specifically. An initial screen was done on duplicate 10-fold dilutions of sera, and if the net absorbance was greater than 0.5, a second assay on additional 3-fold serial dilutions was performed. The ELISA titer was defined as the product of absorbance and dilution factor, when the absorbance is between 0.1 and 0.5. The limit of sensitivity of the IgG assay is 1 unit/ml, with 10 units/ml being a definite positive. The background and variability of the IgM assay is about 3-fold higher. Measurements of IL-2-binding capacity of patients' sera containing IL-2-specifïcIgG indicate that 1 ELISA unit corre sponds to 3 ng/ml of IL-2, or 30 ng/ml of IgG. Since the detection of IgG has no quantitative relation to the identity of the antigen, as long as it is in excess, 1 ELISA unit/ml will correspond to about 30 ng/ml of IgG for any antigen. Sera from patients on trial for 60 days or longer and having an IgG titer of 10 or greater were assayed for ability to neutralize IL-2 activity. IL-2 was added to 3 aliquots of sera to obtain titers of 4000, 400, and 40 units/ml. After incubation at 4°Covernight, the mixture was assayed for IL-2 activity. The neutralizing titer is defined as the fold reduction of IL-2 activity (over the value observed in control sera) times the final serum dilution in the assay. The activity of IFN was measured using an antiviral assay with a visual cytopathic end point (27). The IFN titer was determined by comparison to cells treated with serial dilutions of an NIH standard IFN-/8 preparation of 100 units/ml. Values are reported as the median of triplicate assays. Cell-mediated Cytotoxicity Assays Heparinized whole blood (20 ml) was collected from all patients entered into the study prior to dosing (i.e., 72 h after the last IL-2 dose) at Weeks 0, 3, and 5 and then monthly until time off study. No IL-2 was present in these specimens, which were kept at room temperature and shipped by overnight delivery to the Cetus Corp. Effector Cell Preparation. Patient and normal peripheral blood mononuclear cells were isolated from heparinized blood samples by Product Descriptions Ficoll-Hypaque density gradient centrifugation. The samples were IL-2 and recombinant IFN-/3 were provided by the Cetus Corp. The mixed (approximately 20 ml) with an equal volume of RPMI 1640, IL-2 had a nominal specific activity of 3 x IO6Cetus units/mg (8 x IO6 carefully layered over 20-25 ml of Ficoll-Hypaque, and centrifuged for Biological Response Modifier Program units/mg) equivalent to 1 mg 30 min at 400 x g. The interface of the plasma and Ficoll-Hypaque of product. The IL-2 was supplied as a lyophilized powder which when was removed, the peripheral blood mononuclear cells were washed reconstituted had a concentration of 3 x IO6 Cetus units/ml. The ß- twice in RPMI 1640 and then resuspended in 20-30 ml of RPMI plus interferon had a nominal specific activity of 1.8 x 10s lU/mg. The 5% human AB+ serum, and the concentration was adjusted to 1 x IO7 IFN-/3 was supplied as a lyophilized cake which when reconstituted peripheral blood mononuclear cells/ml. yielded 45 x IO6 lU/ml. Target Cell Preparation. A subclone of the natural killer cell-resistant Daudi human lymphoblastoid cell line was used as the target to evaluate IL-2 Pharmacokinetics LAK activity. Target cells were labeled by a 2-h incubation of 2-4 x IO6cells in 0.5 ml of culture medium supplemented with 200-400 pCi Serum samples were collected in 10-ml serum separator tubes (Becof 51Cr (250-500 ¿iCi/mg;Amersham Corp., Arlington Heights, IL). ton-Dickinson) from all patients entered into the study at 1, 5, 10, 30, Cells were washed three times, with gentle resuspension after pelleting, 60, 120, 240, and 360 min following their initial IL-2 and IFN-0 and then adjusted to 2 x IO5 cells/ml. One hundred /¿I (2 x IO4 cells) dosing. The specimens were processed by centrifugation and pipeted off into cryotubes. The serum samples were then frozen at —40°C until of each cell type were counted in a Beckman gamma counter with a 51Cr isoset to determine the level of 5lCr uptake; 10,000-20,000 cpm/ they were shipped overnight on dry ice to the Cetus Corp. for assay. IO4cells was a standard value. Serum levels were determined by a biological assay which has been Four-H "Cr Release Assay. Labeled target cells (2 x 10" cells in 100 described (24). Essentially, IL-2 levels were determined by measuring /¿I) were added to 96-well, round bottomed microtiter plates. Effector the incorporation of tritiated thymidine by an IL-2-dependent cell line (HT2). The serum was heated at 50°Cfor 30 min before the assay to cells were added in a volume of 100 pi, at ratios of effector to target cells of 50:1, 17:1, and 6:1. Each assay plate also included target cells inactivate serum components which are toxic to the HT2 cells. The incubated in medium alone and target cells incubated in medium sensitivity of the assay is usually 0.3 unit/ml (in the serum sample). containing 0.5% Nonidet P-40 detergent. All assay points were done in One unit/ml is defined relative to an IL-2 standard and produces triplicate. After a 4-h incubation at 37°C,the microtiter plates were approximately 50% of maximum incorporation in the well containing the test cells. One Cetus unit has been determined to be equal to 2.3 centrifuged at 500 x g for 3 min. The supernatant fluids were removed with a Skatron harvester (Skatron, Inc., Sterling, VA) and counted in units as defined by the IL-2 standard supplied by the NIH Biological a gamma counter. The results were quantitated as Response Modifier Program. Assay for Anti-IL-2 Antibodies % of specific lysis = Serum samples were collected in 10-ml serum separator tubes (Becton-Dickinson) from all patients entered into the study prior to dosing at Weeks 0, 3, and 5 and then monthly until they went off study. The serum was separated and then frozen at —40°C. A standard indirect test cpm - spontaneous cpm — x 100% total cpm —spontaneous cpm Spontaneous cpm were defined by the target cells incubated alone; total cpm were defined by target cells in medium plus detergent. Results were calculated in lytic units30%/107effector cells (28). 3876 Downloaded from cancerres.aacrjournals.org on June 15, 2017. © 1988 American Association for Cancer Research. PHASE I STUDY OF IL-2 AND IFN-0 •y-Interferon Assay Serum samples were collected with the pharmacokinetic samples in 10-ml serum separator tubes (Becton-Dickinson) from all patients entered into the study at 2, 60, 120, 240, and 360 min following the initial IL-2 and IFN-/3 dosing. The serum was separated, then frozen at -70°Cuntil assayed. IFN-7 levels were determined using the IMRX 7-interferon radioimmunoassay test kit (Centocor, Inc.). One anti-IFN7 mouse monoclonal antibody-coated polystyrene bead was placed into each well of a 25-well reaction tray. Pipeted into the wells was 0.2 ml of the following: (a) normal human serum standards; (/)) positive control; and (c) specimen sample. The product was then incubated for 2 h at room temperature. Following incubation the liquid was aspirated and the beads were washed three times with a total of 3 ml of distilled water, '"¡-labeled anti-IFN-7 antibody (0.2 ml) was then pipeted onto all of the beads and incubated 2 h at room temperature. The liquid was again aspirated and the beads were washed as before. The beads were then transferred to counting tubes placed in a gamma scintillation counter. RESULTS Patient Characteristics. After obtaining informed consent, 47 patients were entered on the clinical trial. All 47 patients were évaluablefor toxicity analysis. Fifteen patients were withdrawn from the study program prior to completing the initial 4-week treatment period. Eight patients developed early evidence of progressive disease, three patients were removed because of development of infection (one with influenza and two with postobstructive pneumonia), and four patients had unusual, early, non-Grade III or IV toxicities (see below). The remaining 32 clinically évaluablepatients (Table 2) ranged in age from 24 to 70 years with a mean age of 53 years and include IS males and 17 females. The patients had the following diagnoses: colorectal carcinoma, 12; non-small cell lung carcinoma, 4; renal cell carcinoma, 4; malignant mela noma, 3; metastatic breast carcinoma, 3; carcinoid, 1; esophageal carcinoma, 1; oat cell lung carcinoma, 1; ovarian carci noma, 1; synovial sarcoma, 1; and adenocarcinoma of unknown primary, 1. Of this population, 26 patients had received prior chemotherapy (including 1 with prior IFN-/3), 10 had received prior radiotherapy, and 1 had received monoclonal antibody therapy. Three patients had received radiotherapy as their only prior treatment modality. Clinical Effects (Dose Escalation). IL-2 was administered s.c. to the first 35 patients entered on the trial. The dose was limited to 5 x IO6 units/m2 because of the small volume which could be administered by this route. In addition, neutralizing antibod ies to the IL-2 developed in five patients (see below). For these Table 2 Characteristics of patients évaluable for both toxicity and response Total Age (years) Sex Diagnoses Colorectal Non-small cell lung cancer Renal Melanoma Breast Other No. 32° 24-70 15 M, 17 F 12 4 4 3 3 6 Prior treatments Chemotherapy 26 Radiotherapy 10 Monoclonal antibody 1 "An additional 15 patients were available for pharmacological toxicity analysis only. and acute reasons IL-2 was given i.v. to subsequent patients entering the study. This is shown in Table 1. With 5 x IO6 units/m2 of IL2 s.c., the maximum tolerated dose for IFN-/3 was 6 x IO6 units/m2. However, with this same dose of IL-2 administered i.v., the maximum tolerated dose for IFN-/3 was 10 x IO6units/ m2. This was based upon the observed cumulative toxicities of fatigue, anorexia with weight loss, depression, and decrease in overall performance status. Four patients were removed or asked to be removed from the study program because of unacceptable side effects. One patient at Level IV developed a progressive, generalized urticaria! rash during Week 1 which may have been treatment related. The second patient was at Dose Level IV; he was 70 years of age with a primary diagnosis of adenocarcinoma of the lung. After each of three treatments he experienced pronounced dyspnea secondary to rigors and complicated by anxiety. The third patient in this subgroup was at Dose Level IV; he was 56 years of age with a primary diagnosis of melanoma. Each of three doses produced a transient fever of 40.6°Cpreceded by rigors of approximately 1-h duration. During the episode of rigors, the patient had 3-4 episodes of vomiting despite standard antiemetic therapy. On Day 8 of treatment the patient was given 50% doses, due to a decrease in performance status, with similar side effects again recorded, and he asked to be with drawn from study. The fourth of these patients was at Dose Level VI; she was 31 years of age with a primary diagnosis of malignant melanoma. Each of 2 doses produced a transient fever of <39.8°Cpreceded by rigors for approximately 40 min. The patient also experienced nausea and a single episode of vomiting during her rigors. The patient deemed the side effects unacceptable and withdrew from the study. The toxicity data on all 47 patients are summarized in Table 3. Pharmacokinetics. The pharmacokinetic curves of IL-2 (Fig. 1) were generated from data compiled on those patients who received 5 x 106/m2 by either s.c. or i.v. administration. One min after i.v. bolus injection detectable serum levels ranged from 2000 to 6100 units/ml with a mean value of 4060 units/ ml. IL-2 given i.v. exhibited biphasic serum clearance kinetics with an initial clearance of approximately 1-3 min followed by a slower phase of approximately 50 min. Administration of IL2 by the s.c. route resulted in peak serum levels of approximately 1-3% of those seen with i.v. administration. These peak levels occurred at 240-360 min following s.c. administration. At 6 h, comparable IL-2 levels for i.v. and s.c. administration ranged from 36 to 110 units/ml and 8.2 to 190 units/ml, respectively. Response Data. Of the 32 évaluablepatients, 27 had meas urable disease and 5 had évaluabledisease, to assess for re sponse. All but one of the patients had progressive disease at time of entry on study. One patient with renal carcinoma had a stable renal mass. Seven patients had progressive disease upon completing 4 weeks of therapy and were removed from study [3 at Dose Level I, 1 at Level II, 1 at Level V (s.c.), 1 at Level VI (i.v.), and 1 at Level VII (s.c.)]. Twenty-one patients had stable disease after Week 4. One patient was removed from study due to Grade IV fever toxicity after 5 weeks of therapy and remains alive. The mean on-study time for patients with stable disease was 101.2 days and ranges from 28 to 252 days. A total of 10 patients remain alive in the SD group (50%). Three patients had a minor response while on therapy. The first of these patients (at Level III) had a carcinoid tumor metastatic to bone and soft tissue. After Week 4 of therapy, his bone scan and soft tissue disease remained stable. After Week 7 of therapy, his soft tissue disease had decreased by 20% and 3877 Downloaded from cancerres.aacrjournals.org on June 15, 2017. © 1988 American Association for Cancer Research. PHASE I STUDY OF IL-2 AND IFN-)3 Table 3 Toxicities ofll.-2 and IFN-ßin combination toxicity*Fatigue/performancestatus1133243Anorexia/wt with Grade III or IV ofpatientsentered/evaluable"4/44/45/37/37/35/33/35/44/43/3No.requireddosereduction0012332243No. DoselevelIIIIIIIVV loss1121332Depression2222Arthritis122OtherIe1"1*''A1*1'y (S.C.)VI (s.c.)VII (s.c.)V (i.V.)VI (i.V.)VII (i.V.)No. °After 4 weeks of treatment. * Note that individual patients are included under more than one toxicity. ' Hypotension. d Exfoliative skin rash. ' Thrombocytopenia. r Diarrhea. * Fever. * Peripheral edema. sH to H 1500 200.0 250.0 4000 TIMK (miniiU-s) JÕL Fig. 1. Pharmacokinetics of IL-2 with i.v. (IV; A) and s.c. (SQ; O) adminis tration at Dose Level V. Dose Level V uses 5x10" units/m2 of IL-2 and 6x10' units/m! of IFN-/3. The area of the IV curve is 25,898 unit-min/ml, and the area of the SQ curve is 17,498 unit-min/ml (P < 0.05). 10,000 100.000 1.000.000 10.000.000 DOSE IL-2 (I'NITS/M2) bone disease continued as stable. After 9 weeks of therapy, the soft tissue response was at 50% reduction, but there was no change in his bone disease. However, at Day 133 he was removed from study due to tumor progression. The second patient [at Level V (s.c.)] had multiple, bilateral lung métastases from her synovial sarcoma. After 4 weeks of therapy, her measurable disease had decreased by 25% and remained stable H until disease progression at on-study Day 63. The third patient Z [at Level V (s.c.)] with malignant melanoma metastatic to the orbit had a stable computed tomography scan after Week 4 of therapy. However, her physical examination demonstrated a marked decrease (to near normal) in the degree of orbital protuberance and complete resolution of pain and inflammation of the surrounding tissues and structures. She was removed from study at Day 31 due to a decrease in performance status. Two of these patients remain alive. One patient with melanoma had a partial response after Week 1,000.000 10,000,000 100.000.000 4 of therapy at Dose Level IV. A measurable, biopsy-proved, DOSE IFN-BETA (UNITS/M1) right neck mass decreased by >50%. At Week 11 of therapy, Fig. 2. A, maximum LAK activity by treatment dose level of s.c. IL-2; constant the neck examination was negative; however, a nonmeasurable dose IFN-/3, 2 x 10' units/m2. B, maximum LAK activity by IFN-/3 dose level; hilar mass remained unchanged. The patient was removed from constant dose IL-2, 5 x 10' units/m2. Bars, SEM. study at Week 38 (Day 270) because of progressive disease. LAK Activity. The mean LAK activity steadily increased as the dose of IL-2 increased. Significant LAK activity required 5 the difference between mean LU for all dose levels using this x IO6 units/m2 (Fig. 2A). With this dose of IL-2,6 x IO6units/ dose of IL-2 was not statistically significant (Fig. 2B). m2 of IFN-/3 generated the highest mean value for LU. However, No patient had baseline LAK activity >8 LU. However, seven Bo SO" rii 3878 Downloaded from cancerres.aacrjournals.org on June 15, 2017. © 1988 American Association for Cancer Research. PHASE I STUDY OF IL-2 AND IFN-0 évaluablepatients had treatment-stimulated peak LAK activity >15 LU (7 of 32 or 21.9%). These patients had a variety of tumors: colon carcinoma, 2 of 12; melanoma, 2 of 3; breast carcinoma, 1 of 3; renal carcinoma, 1 of 4; and ovarian carci noma, 1 of 1. Of the 12 évaluablecolorectal cancer patients, 3 had PD at Week 4, and 9 had SD. Of these 9 patients, 7 did not exhibit significant LAK activity and had a mean duration of disease stabilization of 87.8 days. Four of these patients are alive. One of the 11 had significant LAK activity at Dose Level V (i.v.), with an on-study time of 58 days. One patient, at Dose Level VII (s.c.), had PD at Week 4 and exhibited significant LAK activity. Both of these patients remain alive to date. Of the 3 évaluablepatients with melanoma, 1 had SD at Week 4 with PD at Week 5. Two patients exhibited significant LAK activity at Levels IV and V (s.c.). One had a marginal response of 31+ days and the second patient had a partial response lasting for 270 days. One patient is alive to date. Of the 3 évaluablepatients with breast cancer, the 1 patient with SD at Week 4 also had significant LAK activity at Level VI (i.v.). This patient had a disease stabilization duration of 71 + days. One patient with metastatic ovarian cancer and SD at Week 4 exhibited significant LAK activity at Dose Level VII (i.v.). This patient elected no further therapy due to severe fatigue at Week 10. Of the 4 évaluablepatients with renal cell carcinoma, 2 had SD including the 1 patient with significant LAK activity with an on-study time of 81 days. Three of these patients remain alive to date. The overall rate of LAK activity >15 LU for the évaluable patients receiving s.c. doses of IL-2 was 18% (4 of 22). The rate of LAK activity >15 LU in the group of évaluablepatients receiving i.v. IL-2 was 30% (3 of 10). Seven of the 21 (33%) évaluablepatients receiving 5 x 10* units/m2 of IL-2 had LAK activity >15 LU, including 4 of 11 (36%) receiving s.c. IL-2. Peak LAK activity occurred at the end of the second week of treatment in 3 patients, at the end of the fourth week of treatment in 2 patients, and at the end of 8 weeks of treatment in 2 patients. There appeared to be a correlation between degree of response and LAK activity (Fig. 3). There was a significant difference in LAK activity between patients having progressive disease (mean, 5.0) and those having either stable disease (mean. !!;/>= 0.047) or a response (mean, 53; P = 0.019). The difference in LAK activity between patients with stable disease and a response was not statistically significant (P = 0.10). Antibody Studies. In order to follow the possible development of antibodies to IL-2 and IFN, an ELISA specific for IgG and for IgM to each of these proteins was performed on serum taken before treatment and at approximately 2-week intervals during their administration. Two or more serum samples were available from 38 of the 47 patients enrolled in this trial. The antibody levels are expressed as titers, defined as the product of the absorbance in the ELISA and the serum dilution factor. A titer of 1 corresponds to approximately 30 ng/ni I of IgG and is the lower limit of the sensitivity of this assay. IgM specific to IL-2 was seen in 12 patients, although the level was only marginally detectable in 7 of these. IgG specific to IL-2 was seen in 24 patients, with the level being marginal in 5 (Fig. 4). In only 2 patients was a n<mmarginal level of IgM not associated with IgG. The maximum IgM titer was 80, while the maximum IgG titer seen was 1600. The four highest IgM titers were found in the group of 12 patients receiving IL-2 by the i.v. route. However, the highest IgG titers were found in patients receiving s.c. injections. There appears to be a signifi cant correlation between maximum IgG titers and number of days on trial only for the group of 26 patients in the s.c. group, as shown in Fig. 4. The sera from all patients with IL-2 antibody titers greater than 10 who received IL-2 for 50 days or longer were assayed for IL-2-neu tral i/ing activity. Among these 9 patients, the sera from 5 had neutralizing activity, with the highest titer being 9000. The immunogenicity of IFN, given by the i.v. route to the entire group, was modest compared to that of IL-2. One patient developed an IFN-specific titer of 660, 3 had titers of 1 or 2, and the remaining patients were negative. Four had non-zero IFN-specific IgM titers, but only in the range of 2-4. Interest ingly, the patient with the IFN antibody titer of 660 also had the highest IL-2 antibody ELISA titer and IL-2-neutralizing activity. 7-Interferon Levels. Endogenous IFN-7 was induced as shown in Fig. 5 following the administration of IL-2 and IFNß.At equivalent doses of IL-2 (5 x 10* units/m2) i.v. adminis tration induced significantly more IFN-7 than did s.c. admin istration (P = 0.047; see legend to Fig. 5). In addition, the maximum induced IFN-7 level occurred 4 h after the IL-2 û= SQ DOSING G = IV DOSING MR/PR n=4 mean-53.10 stm =35 61 >1000- •Z <1000- •f. < 2 H > D D Q D K < 100-1 D G û û CO a = ° t. O 01 0- SD n-21 D A û in =fi 02 n=2.96 <20 <80 DAYS Fig. 3. Mean peak LAK activity according to response. There is a significant difference in LAK activity between patients with progressive disease (PD) and those with either stable disease (SD) (P = 0.047) or a response (P = 0.019). MR. minor response; PR. partial response. The difference in LAK activity between patients with stable disease and a response is not statistically significant (/' = 0.10). Bars, SEM. Fig. 4. Maximum IgG titers in 26 patients receiving i.v. (/r. D) or s.c. (SQ. A) IL-2. The number of days is divided into 4 groups and the IgG titers into 5 groups. There is a significant correlation between maximum IgG titers and number of days on trial for the SQ group (Spearman's p rank correlation, O.S4). There is no correlation between titer and number of days on study for the IV group (Spearman's /) rank correlation. —0.43). 3879 Downloaded from cancerres.aacrjournals.org on June 15, 2017. © 1988 American Association for Cancer Research. PHASE I STUDY OF IL-2 AND IFN-0 Table 4 Toxicities Acute Fever Rigors Vomiting Local inflammation' Diarrhea Rash Chronic Fatigue/performance Anorexia/wt loss" Arthritis/arthralgias" Depression" Peripheral edema" status" Hypotension Insomnia Anemia Thrombocytopenia " The maximum tolerated dose was determined by these chronic toxicities (Grade III-IV). ' After S.C.IL-2. o.o r>o.o loo.o 150.0 200.0 ','fjo.o 300.0 350.0 400.0 TIMK (iniiiuli's) Fig. 5. Endogenous IFN-i following 5x10' units/m2 of IL-2 either i.v. (D) or s.c. (A). Time points, means of 12 patient values. Linear regression was used to test if the s.c. and i.v. routes were equivalent. Having rejected this (P = 0.047), the data were fit by functions of the form a(/)(f"/>)exp(—b*t), i —1, 2 for this graph. The ratio of the two curves is thus constant over time since they differ only in the coefficients a(i). The i.v. curve is uniformly about 3.3 times higher than the s.c. curve. With these fitted curves, the P value is much lower (P = 0.001). administration, with a sharp rise between 2 and 4 h and a gradual decline thereafter. DISCUSSION Initial enthusiasm followed the first reports on the use of interleukin 2 in humans (1, 2). However, significant toxicity was associated with this therapy, and subsequent reports mini mized the potential benefit of this new treatment (29-31). Phase II studies of IL-2 have shown efficacy in renal cell carcinoma and malignant melanoma (3, 4, 8, 32). These two diseases are also responsive to the type I interferons, a- and /3-interferons have reported response rates between 10 and 30% in renal cell carcinoma and between 10 and 20% in malignant melanoma (33, 34). The combination of IL-2 and IFN-/3 was prompted not only by these clinical data but also by preclinical in vitro data demonstrating synergism between these two agents both in stimulating NK activity and in increasing expression of IL-2 receptors (13-18). Interleukin 2 also generates IFN--y in vitro, a substance that is synergistic both with type I interferons and with interleukin 2 (35). IFN--y has been shown to generate IL2 receptors and thereby presumably enhance IL-2 binding (36, 37). In addition, IFN-7 binds to a different receptor than do the type I interferons, subsequently enhancing the antiproliferative effects of each other (21-23). In the current study, IL-2 and IFN-/3 were given by i.v. bolus, three times weekly. The doses of interleukin 2 were comparable to those given with interleukin 2 alone in a similar schedule.3 Over a 4-week period of time, 60 million units/m2 of interleukin 2 are administered using this regimen. The IL-2 plus IFN-0 regimen has been shown to be well tolerated and with somewhat different toxicities than are seen with the high dose therapy used by Rosenberg (3). Specifically, the dose-limiting toxicities were the chronic toxicities of profound fatigue, lethargy, depres sion, weight loss, and a decrease in performance status (Table 4). Hypotension, peripheral edema, and pulmonary edema, the major dose-limiting toxicities reported in high dose IL-2 or IL2 plus LAK studies, were uncommon in this moderate dose, intermittent IL-2 plus IFN-/3 trial. This combination was able to generate LAK activity with the 3 E. Hersh, personal communication, 1987. interleukin 2 administered both s.c. and i.v. However, the s.c. dosing induced neutralizing antibodies. There was a 90% inci dence of antibodies when patients were treated s.c. for more than 50 days and 5 of 9 of these were neutralizing. No neutral izing antibodies were seen with the i.v. dosing of IL-2. More over, there was no change in the generation of IL-2 antibodies formed by the addition of IFN-0. Of particular note is that the one patient with a partial response had significant levels of LAK activity. This LAK activity disappeared when she devel oped neutralizing antibodies to the IL-2. As typified by this patient, however, the ability to generate LAK was correlated with response to treatment (Fig. 3). As the degree of response improved from progression to stable disease to minor or partial response, the mean level of LAK activity increased as well. While there was not a statistical difference between the LAK activity in patients with stable disease as compared to those with a response, the trend toward improvement is apparent. The use of i.v. IL-2 resulted in much higher serum levels of interleukin 2 for approximately 3 h as compared to the use of s.c. IL-2. This may account for the difference in the ability to generate 7-interferon between these two routes of administra tion. Attempts are now ongoing to minimize the toxicity seen with high doses of IL-2 and to assess the contribution to efficacy of the administration of LAK. Recent reports have challenged the need for the ex vivo activation of LAK (32, 38, 39). However, most data support the importance of LAK as the mechanism of activity for interleukin 2. Attempts to minimize toxicity and maintain efficacy have included administering the interleukin 2 by continuous infusion, using a lower dose of interleukin 2, and using the same dose of interleukin 2 without the coadministration of LAK. Others have reported on the use of interleukin 2 i.p., s.c., and intermittently i.v. (38-40). The current report presents a novel approach to potentially enhancing the efficacy of moderate dose IL-2 by combining IL-2 with low dose IFN0. The recommended dose for Phase II studies is 5 x 10' units/ m2 IL-2 plus 6 x IO6 units/m2 IFN-/3, both administered i.v. This dose is one level below the maximum tolerated dose and would appear to be an adequate dose for generating LAK activity by this schedule. The use of IL-2 i.v. also avoids the problem of neutralizing antibodies seen with IL-2 s.c. One patient with malignant melanoma had a partial tumor regres sion and one patient had a minor tumor regression. Thus melanoma would appear to be a likely tumor type for Phase II testing. Both IL-2 and IFN-/3 have independent activity in renal cell carcinoma, and therefore this tumor type might also be an appropriate candidate for Phase II studies. There is also the potential for this combination to have synergy with the induced IFN-y. 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A Phase I Study of Recombinant Interleukin 2 plus Recombinant β-Interferon Robert L. Krigel, Kristin A. Padavic-Shaller, Alfred R. Rudolph, et al. Cancer Res 1988;48:3875-3881. Updated version E-mail alerts Reprints and Subscriptions Permissions Access the most recent version of this article at: http://cancerres.aacrjournals.org/content/48/13/3875 Sign up to receive free email-alerts related to this article or journal. To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department at [email protected]. To request permission to re-use all or part of this article, contact the AACR Publications Department at [email protected]. Downloaded from cancerres.aacrjournals.org on June 15, 2017. © 1988 American Association for Cancer Research.