1678 Vol. 6, 1678 –1692, May 2000
`
`Clinical Cancer Research
`
`Phase I Trial of Twice-Weekly Intravenous Interleukin 12 in
`Patients with Metastatic Renal Cell Cancer or Malignant
`Melanoma: Ability to Maintain IFN-g Induction Is
`Associated with Clinical Response1
`
`Jared A. Gollob,2 James W. Mier,
`Korina Veenstra, David F. McDermott,
`Daniel Clancy, Marguerite Clancy, and
`Michael B. Atkins
`Beth Israel Deaconess Medical Center, Division of
`Hematology/Oncology, Boston, Massachusetts 02215
`
`ABSTRACT
`The aim of this study was to examine the tolerability,
`antitumor activity, and biological effects of a new schedule
`of i.v. recombinant human interleukin 12 (rhIL-12). Twenty-
`eight patients were enrolled in a Phase I trial in which
`rhIL-12 was administered twice weekly as an i.v. bolus for 6
`weeks. Stable or responding patients were eligible to receive
`additional 6-week cycles until there was no evidence of
`disease or until tumor progression. Patient cohorts were
`treated with escalating doses of rhIL-12 (30 –700 ng/kg). The
`maximum tolerated dose (MTD) was 500 ng/kg, with dose-
`limiting toxicities consisting of elevated hepatic transami-
`nases and cytopenias. At the MTD (n 5 14), there was one
`partial response occurring after 6 cycles of rhIL-12 in a
`patient with renal cell cancer. Two additional renal cell
`cancer patients treated at the MTD had prolonged disease
`stabilization, with one of these exhibiting tumor regression
`after 8 cycles of rhIL-12. IFN-g, IL-15, and IL-18 were
`induced in patients treated with rhIL-12. Whereas IFN-g
`and IL-15 induction were attenuated midway through the
`first cycle in patients with disease progression, those patients
`with tumor regression or prolonged disease stabilization
`were able to maintain IFN-g, IL-15, and IL-18 induction.
`The down-modulation of IFN-g induction during rhIL-12
`treatment did not relate to IL-10 production or alterations
`in rhIL-12 bioavailability but was associated with an ac-
`quired defect in lymphocyte IFN-g production in response
`to IL-12, IL-2, or IL-15. This defect could be partially
`
`Received 12/20/99; revised 2/18/00; accepted 2/18/00.
`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.
`1 Supported in part by NIH Grants CA78055 and CA74401 as well as by
`a stipend from Genetics Institute, Inc.
`2 To whom requests for reprints should be addressed, at Division of
`Hematology/Oncology, Beth Israel Deaconess Medical Center, 330
`Brookline Avenue, East Campus/Room KS-158, Boston, MA 02215.
`Phone: (617) 667-1930; Fax: (617) 975-8030.
`
`overcome in vitro through combined stimulation with IL-12
`plus IL-2. These findings show that the chronic administra-
`tion of twice-weekly i.v. rhIL-12 is well-tolerated, stimulates
`the production of IL-12 costimulatory cytokines and IFN-g,
`and can induce delayed tumor regression. Strategies aimed
`at maintaining IFN-g induction, such as the addition of IL-2,
`may further augment the response rate to this schedule of
`rhIL-12.
`
`INTRODUCTION
`IL3-12 is a cytokine with considerable promise for the
`treatment of human malignancies because of its pleiotropic
`immunostimulatory effects on lymphocytes (1–5), dendritic
`cells (6), and neutrophils (7– 8), as well as its potent antitumor
`activity in murine tumor models (9 –10). Whereas immune ac-
`tivation by IL-12 in mice has resulted in both tumor necrosis
`factor and NO production, the antitumor effect of IL-12 has
`been more dependent on IFN-g production (10) and the activa-
`tion of either CD81 T cells (9 –11) or NKT cells (12). There
`seem to be a number of mechanisms through which IL-12 can
`induce tumor regression, including the direct killing of tumor
`cells by activated lymphocytes, the antiangiogenic effects of
`IL-12-induced IFN-g (13), and injury both to the tumor micro-
`circulation and to the tumor itself by activated neutrophils (11).
`The immunomodulatory activity of IL-12 is considerably
`dependent on costimulatory cytokines. When the ability of
`IL-12 to activate unmanipulated peripheral blood NK cells and
`CD81 T cells in humans was examined, it was found that these
`lymphocyte subsets responded to IL-12 only when stimulated
`together with IL-2 (14). Both IL-15 and IL-18 are also key
`costimulatory cytokines, which, when combined with IL-12,
`induce strong IFN-g production by T and NK cells (15, 16). In
`mice treated with IL-12, the neutralization of endogenous IL-18
`significantly blunts IFN-g production (17), further emphasizing
`the fact that the biological activity of IL-12 in vivo is likely
`dependent on the presence and/or induction of endogenous
`costimulatory cytokines.
`The promising preclinical data showing IL-12 to be highly
`effective against murine melanoma, renal cell cancer, and sar-
`coma led to its testing in clinical trials in cancer patients starting
`in 1994. In the first published trial, rhIL-12 was administered
`i.v. daily for 5 days, with a 2-week break between cycles. In
`
`3 The abbreviations used are: IL, interleukin; rh, recombinant human;
`NO, nitric oxide; DLT, dose-limiting toxicity; MTD, maximal tolerated
`dose; PR, partial response; PBMC, peripheral blood mononuclear cell;
`CT, computed tomography; NK, natural killer; ppb, parts per billion.
`
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`Clinical Cancer Research
`
`1679
`
`addition, a single test dose was given 2 weeks before the first
`cycle. With that dosing schedule, the MTD was 500 ng/kg, with
`DLTs consisting of liver function test abnormalities and stoma-
`titis (18). Although signs of immune activation were observed,
`including dose-dependent IFN-g production and reversible de-
`creases in CD81 T cell and NK cell numbers (19), only two
`responses were seen among 40 patients (one PR in a patient with
`renal cell cancer and one transient complete response (CR) in a
`patient with melanoma). Similarly low response rates were
`observed in two subsequent trials of weekly s.c. rhIL-12 in
`melanoma (20) and renal cell cancer (21), as well as in a trial
`testing a thrice weekly schedule of s.c. rhIL-12 (22).
`In patients treated with either i.v. or s.c. rhIL-12, IFN-g
`production induced in vivo by rhIL-12 has attenuated rapidly
`with consecutive cycles (18, 20 –22), which indicates that the
`biological response to rhIL-12 is down-modulated during ther-
`apy. Even a single test dose administered 2 weeks before the
`first cycle of rhIL-12 seemed to attenuate IL-12-induced IFN-g
`production (23). This down-modulation of IFN-g production has
`been shown to result in diminished IL-12-induced tumor regres-
`sion in mice (24). In addition, multiple doses of IL-12 have also
`been shown in animals to induce a temporary state of immuno-
`suppression (25–26), perhaps analogous to the down-modula-
`tion of IFN-g production in patients receiving multiple doses of
`rhIL-12. This paradoxical immunosuppression after a relatively
`brief period of immune activation by rhIL-12 may explain the
`limited antitumor activity observed to date in rhIL-12 clinical
`trials. Although the mechanism of this IL-12-induced down-
`modulation of subsequent IFN-g induction remains undefined,
`data from animal models have suggested that IL-12-induced NO
`may be operative (26), whereas observations from clinical trials
`have also implicated changes in rhIL-12 pharmacokinetics
`(20, 22).
`In June of 1998, we initiated a Phase I dose escalation trial
`of i.v. rhIL-12 in patients with renal cell cancer and melanoma,
`using a new dosing schedule. To try to prevent or delay the
`dampening of IFN-g induction, we eliminated the test dose. In
`addition, we implemented a twice-weekly dosing schedule to
`determine whether moderate and sustained IFN-g production
`could be stimulated without prohibitive toxicity. Although im-
`portant aims of this trial included determining the safety and
`tolerability as well as the antitumor activity of this regimen, this
`study was also undertaken to further explore the mechanism
`through which rhIL-12 activates the immune system in vivo and
`to examine how IFN-g induction by rhIL-12 is modulated with
`chronic dosing.
`
`PATIENTS AND METHODS
`Patient Selection. All of the patients were adults with
`histologically proven advanced malignancy that was metastatic
`or unresectable and for which standard curative or palliative
`measures did not exist or were no longer effective. All of the
`patients had measurable or evaluable disease that was clearly
`progressive. Patients were required to have an Eastern Cooper-
`ative Oncology Group (ECOG) performance status of 0 or 1 and
`adequate organ function defined by WBC .4000/ml, platelet
`count .100,000/ml, creatinine ,1.5 mg/dl, bilirubin ,1.5 mg/
`dl, aspartate aminotransferase ,2 times the upper limit of nor-
`
`mal, and electrocardiogram and chest X-ray without clinically
`significant nonmalignant abnormalities. Patients with brain me-
`tastases, seizure disorders, organ allografts, concurrent require-
`ment for corticosteroids, more than two prior chemotherapy
`regimens, more than two prior immunotherapy regimens, or
`prior IL-12 therapy were ineligible.
`Study Design. The study was an open-label, nonrandom-
`ized, single-center Phase I dose escalation trial. The treatment
`protocol was approved by the Cancer Therapy Evaluation Pro-
`gram (CTEP) of the National Cancer Institute (protocol T97-
`0053) and by the Human Institutional Review Board at the Beth
`Israel Deaconess Medical Center (protocol 97-1083), and writ-
`ten informed consent was obtained from each patient. rhIL-12,
`produced by Genetics Institute, Inc. (Cambridge, MA), was
`supplied by the National Cancer Institute (IND 6798). The
`rhIL-12 was administered by i.v. bolus injection.
`The treatment schedule is shown in Fig. 1. Patients were
`treated in the General Clinical Research Center at the Beth Israel
`Deaconess Medical Center, and received i.v. bolus injections of
`rhIL-12 twice weekly, with doses given 3– 4 days apart. A cycle
`of therapy lasted 6 weeks, with patients receiving a total of 12
`doses during that period. During the first cycle only, patients
`were admitted overnight after the first, second, and seventh
`doses of rhIL-12 for observation and serial blood draws. All of
`the remaining doses were administered on an outpatient basis,
`with patients observed for 1 h after each dose. Patients were
`evaluated for tumor response at the end of each 6-week cycle,
`and patients with stable or regressing disease could continue
`receiving additional cycles until there was no evidence of dis-
`ease or until there was disease progression. Patients were al-
`lowed up to a 2-week break between cycles for the resolution of
`any significant rhIL-12-induced toxicity.
`The rhIL-12 dose was increased from 30 to 700 ng/kg in
`successive cohorts of patients. No intrapatient dose escalation
`was permitted. A minimum of three patients were enrolled at
`each dose level, and all of the patients had to have completed the
`first 3 weeks of cycle 1 before initiating enrollment to the next
`dose level. Toxicity was assessed using the National Cancer
`Institute Common Toxicity Criteria. In general, grade 3 or
`greater toxicities were considered dose-limiting. However, liver
`function test abnormalities were not classified as dose-limiting
`until the total bilirubin was .3 times normal or the hepatic
`transaminases or alkaline phosphatase were .10 times normal.
`In addition, the WBC count and neutrophil count were not
`considered dose-limiting until criteria for grade 4 toxicity were
`met, and no degree of lymphopenia was dose-limiting. Grade 2
`cardiovascular toxicity (except for hypotension) and neurolog-
`ical toxicity were considered dose-limiting. The IL-12 dose was
`escalated when 0 of 3 patients at a dose level had a DLT. If 1 of
`3 experienced a DLT, three more patients were enrolled at that
`dose level, and the dose was escalated if no more than 1 of 6
`patients had a DLT. Patients experiencing a DLT could resume
`the IL-12 at the next lowest dose level if the toxicity resolved
`within 2 weeks. When two or more DLTs were experienced at
`a dose level, the MTD was determined to be the next previous
`dose level.
`All of the patients received ranitidine for the duration of
`their IL-12 treatment. Acetaminophen was administered prophy-
`lactically for 24 h after each IL-12 dose and could be taken as
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`1680 Phase I Trial of Twice-Weekly i.v. rhIL-12
`
`X
`
`X
`
`l l l l
`
`Week
`
`1
`
`2
`
`X
`
`l l
`
`3
`
`I
`
`4
`
`l l
`
`5
`
`6
`
`= IL-12 dose administered by IV bolus
`twice weekly on days 1 and 4
`
`trial of
`Fig. 1 Schema for clinical
`twice-weekly i.v. rhIL-12 without a
`test dose.
`
`X = Overnight hospitalizations for serial q4hr blood
`draws to collect serum for measurement of cytokines
`induced following IL-12 injection
`
`•IL-12 dose levels: 30,100,300, 500, 700 ng/kg
`•3-6 patients per dose cohort
`•Tumor response assessed after each 6-week cycle
`•Patients can continue to recehe 6-week cycles until
`no evidence of di,ease or until disease progression
`
`needed thereafter. Indomethacin was used to control fever that
`was not responsive to acetaminophen, and demerol was used to
`treat rigors.
`Assessment of Tumor Response. Tumor measurements
`were obtained by CT scan at the end of each 6-week cycle
`of IL-12.
`Measurement of IL-12- and rhIL-12-Induced Cyto-
`kines. Serial blood specimens were collected in heparinized
`tubes immediately before and 4, 8, 12, 16, 20, and 24 h after the
`first, second, and seventh rhIL-12 doses during the first cycle.
`The tubes were centrifuged immediately after collection, and the
`plasma was then removed and stored at 220°C. Plasma IL-12
`levels were measured using an ELISA that detects only the p70
`IL-12 heterodimer (Endogen, Cambridge, MA, sensitivity ,3
`pg/ml). ELISA kits were also used to measure plasma IFN-g
`(Endogen, sensitivity ,2 pg/ml), IL-10 (Endogen, sensitivity
`,3 pg/ml), IL-15 (R&D, Minneapolis, MN, sensitivity ,1
`pg/ml), and IL-18 (R&D, sensitivity ,15 pg/ml).
`In Vitro Assays of Lymphocyte Cytokine Responsive-
`ness. Blood specimens were collected in heparinized tubes
`immediately before the first and seventh doses of rhIL-12
`during cycle 1. PBMCs were isolated from blood samples
`through density gradient centrifugation using Histopaque-
`1077 (Sigma, St. Louis, MO). PBMCs were incubated in
`96-well U-bottomed plates at 5 3 104 cells/well with medium
`alone (RPMI 1640 plus 15% FCS, 2% L-glutamine, 1%
`sodium pyruvate, 1% gentamicin, and 1% penicillin-strepto-
`mycin) or with medium plus one of the following: (a)
`50 ng/ml IL-2 (Chiron Corporation, Emeryville, CA, specific
`activity 18 3 106 units/mg); (b) 1 nM IL-12 (Genetics Insti-
`tute, Cambridge, MA, specific activity 1.7 3 107 units/mg);
`(c) 10 ng/ml IL-15 (Endogen, specific activity $2 3 106
`units/mg); (d) IL-2 1 IL-12; or (e) IL-15 1 IL-12. Condi-
`tions were plated in triplicate, and after a 72-h incubation at
`37°C, aliquots of supernatants from each well were harvested
`
`immediately before pulsing each well with 1 mCi [3H]thymi-
`dine (DuPont-New England Nuclear, Boston, MA). The
`IFN-g concentration in the harvested supernatants was as-
`sayed using an IFN-g ELISA (Endogen). Cell proliferation
`was determined by measuring [3H]thymidine incorporation
`8 h after pulsing, as described previously (27).
`Measurement of NO in Expired Air. The concentration
`of NO in expired air was measured in five patients receiving
`IL-12 at either the 500-ng/kg or the 700-ng/kg dose levels.
`Measurements were made immediately before and 24 h after the
`first and second IL-12 doses. Expired air was collected in
`self-sealing balloons after first clearing the upper airway of
`ambient air NO by having patients take four deep inspirations
`through a tube fitted with a charcoal filter (Omega Engineering
`Co.). The NO concentration in the air expired after the fourth
`breath was measured using a high-sensitivity NO detector based
`on a gas-phase chemiluminescent reaction between NO and
`ozone (Model 280 Nitric Oxide Analyzer, Sievers Instruments,
`Inc., Boulder, CO). Patients receiving high-dose IL-2 (600,000
`IU/kg i.v. every 8 h) were used as positive controls. IL-2
`patients had NO samples obtained before the start of the 1st
`week of IL-2 and then daily for the 1st 3 days of IL-2 treatment.
`
`RESULTS
`Patient Characteristics
`Between June 1998 and June 1999, 28 patients were en-
`rolled in this study. Patient characteristics are shown in Table 1.
`The majority of patients had metastases to two or more sites
`(including 15 of 28 with liver, adrenal, and/or kidney involve-
`ment and 10 of 28 with bone metastases), and 23 of 28 had
`received one or more prior immunotherapy regimens (primarily
`IL-2-based regimens). Only 3 of 23 patients had responded to
`their prior immunotherapy.
`
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`Table 1 Patient characteristics
`
`Total patients
`Median age (range)
`Gender (male/female)
`Performance status
`ECOGa 0
`ECOG 1
`Tumor types
`Renal cell cancer
`Melanoma
`Prior therapy
`Chemo/Immunotherapy
`High-dose IL-2
`Low-dose IL-2
`IFN a-2b
`Biochemotherapy
`Chemotherapy
`Surgery
`Radiotherapy
`None
`Prior systemic treatment regimens
`0
`1
`2
`3
`Prior response to chemo/immunotherapy
`Disease sites
`Lung
`Lymph nodes
`Skin or soft tissue
`Liver
`Bone
`Adrenal/kidney
`Number of disease sites
`1
`2
`$3
`a ECOG, Eastern Cooperative Oncology Group.
`
`No. of patients
`28
`56 yr (32–72 yr)
`20/8
`
`15
`13
`
`20
`8
`
`24
`14
`6
`12
`3
`1
`20
`13
`1
`
`4
`12
`10
`2
`3
`
`21
`13
`5
`9
`10
`6
`
`6
`9
`13
`
`Treatment Administered and Toxicity
`Dose Escalation Phase. Three patients were treated at
`each of the first three rhIL-12 dose levels (30, 100, and 300
`ng/kg), with no DLTs. Common side effects included self-
`limited fever and chills, occurring 6 –10 h after the dose, and
`mild malaise. These side effects were observed even at the
`30-ng/kg dose level, and were greatly attenuated by the start of
`the 3rd week of therapy. One patient, treated at 100 ng/kg,
`developed a supraventricular tachycardia (Table 2) in the setting
`of fever after the second rhIL-12 dose, which resolved sponta-
`neously. Small, minimally symptomatic, transient oral aphthous
`ulcers developed during the first cycle of therapy in one patient
`at the 300-ng/kg dose level. No significant (Grade 2 or greater)
`cytopenias or liver-function test abnormalities were noted at the
`first three dose levels.
`At the 500-ng/kg dose level, the fever and chills were more
`severe with previously untreated patients or with patients for
`whom more than 1 year had passed since prior therapy. Fevers
`were highest after the second dose and were minimal-to-absent
`by the 3rd week of therapy in the majority of patients. Indo-
`methacin was added to acetaminophen for the control of fevers
`and chills in only 3 of 14 patients and was never required
`beyond the first week of therapy. Minimal nausea and anorexia
`
`Clinical Cancer Research
`
`1681
`
`were observed, but no diarrhea or gastrointestinal bleeding.
`Stomatitis was uncommon and was never greater than grade 2.
`Grade 1–2 elevations of serum transaminases were common,
`usually peaking after the second dose and normalizing by the
`start of week 3 (Table 2). Orthostatic hypotension 24 h after
`the second rhIL-12 dose occurred in one patient and constituted
`the one DLT among the six patients treated at the 500-ng/kg
`dose level during the escalation phase. No fluid retention or
`evidence of capillary leak syndrome was observed at either the
`500-ng/kg dose level or any other rhIL-12 dose level, nor was
`there any renal or pulmonary toxicity.
`A total of five patients were treated at the 700-ng/kg dose
`level. Two patients (one with melanoma and one with renal cell
`cancer) who had received high-dose IL-2 ,6 months before the
`rhIL-12 had either no fever or low-grade fevers and minimal-
`to-no liver function test abnormalities during rhIL-12 treatment.
`In contrast, the other three patients who received either high-
`dose IL-2 therapy .1 year previously or low-dose IL-2 .6
`months previously experienced higher and more sustained fe-
`vers (requiring both acetaminophen and indomethacin during
`the first 2 weeks of therapy) as well as more protracted consti-
`tutional symptoms (including malaise and anorexia). Two DLTs
`were observed among these three patients, including grade-3
`hemolytic anemia (occurring during week 5 of cycle 1) in one
`patient and a grade-3 elevation of serum hepatic transaminases
`(occurring after the second dose of rhIL-12) in another (Table
`2). The hemolytic anemia was Coombs negative and required
`both the discontinuation of the rhIL-12 and a 1-week course of
`prednisone to resolve. IL-12-induced hypersplenism leading to
`extravascular hemolysis was suspected because CT scans
`showed the development of splenomegaly after the first cycle of
`rhIL-12 (not shown). The grade 3 transaminase elevation re-
`solved within 1 week of stopping the rhIL-12.
`Safety Phase. On the basis of the two DLTs observed at
`the 700-ng/kg dose level, the MTD for the twice-weekly sched-
`ule of i.v. rhIL-12 administered without a test dose was deter-
`mined to be 500 ng/kg. To better assess the safety of the MTD,
`an additional eight patients were treated at 500 ng/kg. As shown
`in Table 3, 7 of 8 patients tolerated the rhIL-12 well without any
`DLTs. One patient tolerated cycle 1 without difficulty but then
`developed grade-4 neutropenia after the first 2 weeks of cycle 2.
`Bone marrow biopsy revealed agranulocytosis, which resolved
`after discontinuation of the IL-12 and treatment with prednisone
`plus low-dose oral cyclophosphamide.
`With the exception of the case of agranulocytosis, no
`unusual or severe toxicities occurred among patients receiving
`two or more uninterrupted 6-week cycles of rhIL-12, including
`two patients who had been on rhIL-12 for 36 and 48 weeks,
`respectively (Table 3). Several patients, including the one on
`rhIL-12 for 36 weeks, experienced grade 1–2 arthralgias (Table
`2), involving primarily the shoulders and fingers, beginning
`with the second cycle of therapy. The arthralgias were episodic,
`unaccompanied by joint swelling or tenderness, and responsive
`to therapy with nonsteroidal anti-inflammatory drugs.
`
`Biological Effects of Twice-Weekly i.v. rhIL-12
`IFN-g levels were obtained in
`In Vivo IFN-g Induction.
`eight patients treated at the 500-ng/kg dose level as well as
`in two patients enrolled at the 700-ng/kg dose level. As shown
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`1682 Phase I Trial of Twice-Weekly i.v. rhIL-12
`
`Table 2 Number of patients experiencing select toxicities during treatment with IL-12 (grades 2, 3, 4)
`
`Dose level, ng/kg (no. of patients)
`
`Toxicity
`
`30 (3)
`
`100 (3)
`
`300 (3)
`
`500 (14)
`
`Hepatic
`ASTa
`Bilirubin
`Alk phosphatase
`Hematologic
`Neutropenia
`Anemia
`Hemolytic anemia
`Thrombocytopenia
`Oral mucositis
`Cardiovascular (hypotension)
`Cardiovascular (arrhythmia)
`Fever
`Arthralgia
`a AST, aspartate aminotransferase; Alk, alkaline.
`
`(1, 0, 0)
`
`(1, 0, 0)
`(1, 0, 0)
`
`(4, 0, 0)
`(1, 0, 0)
`(1, 0, 0)
`
`(2, 1, 1)
`(2, 0, 0)
`
`(1, 0, 0)
`(2, 0, 0)
`(1, 0, 0)
`
`(2, 1, 0)
`(2, 0, 0)
`
`700 (5)
`
`(0, 1, 0)
`
`(1, 0, 0)
`
`(2, 0, 0)
`
`(0, 1, 0)
`(1, 0, 0)
`(1, 0, 0)
`
`(2, 0, 0)
`
`Table 3 Summary of tolerability of IL-12 among patients treated at the MTD of 500 ng/kg
`
`Dose escalation phase
`
`Safety phase
`
`Patient
`no.
`10
`11
`12
`
`13
`14
`15
`21
`22
`
`23
`24
`25
`26
`27
`28
`
`No. of
`cycles
`completed
`8
`3
`1
`
`6
`1
`1
`1
`2a
`
`1
`2
`1b
`1
`3
`2
`
`Results
`
`No DLT
`No DLT
`Grade 2 orthostatic hypotension; IL-12
`dose reduced to 300 ng/kg
`No DLT
`No DLT
`No DLT
`No DLT
`Grade 4 neutropenia (agranulocytosis);
`IL-12 discontinued
`No DLT
`No DLT
`No DLT
`No DLT
`No DLT
`No DLT
`
`a Received four doses in cycle 2.
`b Received only two doses in cycle 1 because of rapid disease progression.
`
`in Fig. 2 and Table 4, we were able to discern three patterns of
`IFN-g induction among these 10 patients. In all of the patterns,
`the first significant rise in plasma IFN-g occurred between 4 and
`8 h after the rhIL-12 dose, corresponding to the onset of fevers/
`chills. In the type-I pattern (Table 4 and Fig. 2A, top), the IFN-g
`level peaked at a modest 450-1600 pg/ml (with peaks occurring
`between 8 and 24 h for individual patients) after the first rhIL-12
`dose (week 1/day 1). After the second dose (week 1/day 4), peak
`levels were 2–3-fold higher than those induced by the first dose.
`However, after the seventh dose (week 4/day 1), peak IFN-g
`levels were comparable with those after the first dose. Patients
`with this type-I pattern tended to have modest fever/chills after
`each rhIL-12 dose during cycle 1, with the most prominent
`symptoms occurring after the second dose. However, whereas
`IFN-g could be detected in the plasma 24 h after an IL-12 dose,
`it always dropped to undetectable levels by the time of the next
`dose 2–3 days later (Fig. 2, A-C, top). Patients exhibiting the
`
`type-I pattern of IFN-g induction had all been treated previously
`with an IL-2-based regimen and were either .6 months past a
`low-dose IL-2 regimen or .1 year past a high-dose IL-2
`regimen.
`In the type-II pattern, peak IFN-g levels after the first dose
`were, on the average, 2-fold higher than those measured in
`patients with the type-I pattern (Table 4 and Fig. 2B, top). The
`augmentation in peak IFN-g levels after the second dose was
`also higher in the type-II pattern compared with the type-I
`pattern, increasing 2- to 4-fold over the peak levels after dose 1.
`This difference in the magnitude of IFN-g production was
`associated with higher fevers and more pronounced chills/rigors
`in these patients after the first two doses of rhIL-12 compared
`with patients exhibiting the type-I pattern of IFN-g induction.
`However, despite this larger surge of IFN-g production after the
`second dose, IFN-g induction after the seventh dose of rhIL-12
`was markedly curtailed compared with IFN-g levels after the
`
`NOVARTIS EXHIBIT 2038
`Breckenridge v. Novartis, IPR 2017-01592
`Page 5 of 15
`
`

`

`A
`
`3000
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`2500
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`Clinical Cancer Research
`
`1683
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`Time (hours post IL-12 injection)
`Fig. 2 Patterns of in vivo cytokine induction in patients during rhIL-12 therapy. Plasma concentrations of IFN-g, IL-15, and IL-18 were measured
`in 10 patients treated with rhIL-12 at the 500-ng/kg and 700-ng/kg dose levels. Cytokine concentrations were determined before and after (every 4 h
`over a 24-h period) the first (Wk1D1), second (Wk1D4), and seventh (Wk4D1) rhIL-12 injections during cycle 1. The values for IFN-g, IL-15, and
`IL-18 at each time point in A represent the mean and SD derived from four patients with the type-I pattern of IFN-g induction. In B and C, representing
`the type-II and type-III patterns of IFN-g induction, respectively, the values at each time point for IFN-g and IL-15 represent the mean and SD derived
`from three patients for each pattern, whereas the data for IL-18 were from one patient (similar results seen with two patients examined) for each
`pattern.
`
`first dose. This was associated with a greatly diminished-to-
`absent febrile response to rhIL-12 by the third week of cycle 1.
`All of the three patients with this pattern of IFN-g induction had
`not received any prior immunotherapy for their metastatic dis-
`ease.
`
`The type-III pattern was characterized by modest peak
`IFN-g levels in response to the first dose of rhIL-12, followed
`by the rapid attenuation of IFN-g production (Table 4 and Fig.
`2C, top). Whereas peak IFN-g levels reached their maximum
`after the second rhIL-12 dose in the type-I and type-II patterns,
`in the type-III pattern, peak IFN-g levels were lower after dose
`2 compared with those after dose 1 (Table 4), and they contin-
`ued to decline when measured again after the seventh dose. This
`type-III pattern was associated with a weak-to-absent febrile
`response to IL-12, and, of the three patients exhibiting this
`pattern, all had received one course or multiple courses of
`high-dose IL-2 ,6 months before starting the rhIL-12.
`
`In Vivo Induction of IL-15 and IL-18. To determine
`whether IL-15 and IL-18 were induced by rhIL-12 in cancer
`patients and to examine whether there was an association be-
`tween IL-15/IL-18 induction and IFN-g induction by rhIL-12,
`we measured the plasma levels of IL-15 and IL-18 at the same
`time points used to measure IFN-g levels. As shown in Fig. 2,
`A-C (middle), IL-15 was not detectable in the plasma before
`starting rhIL-12 but was detectable at low levels 4 h after the
`first injection. With each pattern of IFN-g induction, plasma
`IL-15 levels reached their maximum either before, or at the
`same time as, peak IFN-g levels. However, the magnitude of
`IL-15 induction did not correlate with the magnitude of IFN-g
`induction, because peak IFN-g levels after the second rhIL-12
`dose in the type-I and type-II patterns were not associated with
`similar increases in peak IL-15 induction. Nonetheless, there
`was an association between the ability to sustain comparable
`levels of IL-15 induction during a cycle of rhIL-12 and the
`
`NOVARTIS EXHIBIT 2038
`Breckenridge v. Novartis, IPR 2017-01592
`Page 6 of 15
`
`

`

`1684 Phase I Trial of Twice-Weekly i.v. rhIL-12
`
`Table 4 Relation between pattern of IFN-g induction by IL-12 during cycle 1, clinical response, and cytopenias
`
`IFN-g
`induction
`pattern
`Type I
`Patient 10
`13
`18
`22
`
`Type II
`Patient 24
`26
`28
`
`Cycle 1 Peak IFN-g level (pg/ml)
`
`Disease
`
`Wk1D1a
`
`Wk1D4
`
`Wk4D1
`
`IL-12 dose
`level
`(ng/kg)
`
`RCC
`RCC
`RCC
`RCC
`
`RCC
`RCC
`Mel
`
`1600
`1100
`450
`1000
`
`2365
`2040
`1560
`
`2560
`2235
`1290
`1840
`
`4960
`9060
`5918
`
`1200
`1340
`400
`850
`
`220
`248
`77
`
`500
`500
`700
`500
`
`500
`500
`500
`
`Response
`
`Cytopenia
`
`SD @48 wk
`PR @36 wk
`SD @20 wk
`SD @24 wk
`
`PD @12 wk
`PD @6 wk
`PD @12 wk
`
`None
`None
`Hemolytic anemia
`Agranulocytosis
`
`None
`None
`None
`
`Type III
`None
`PD @6 wk
`500
`159
`1476
`2229
`Mel
`Patient 21
`None
`PD @6 wk
`500
`90
`951
`1256
`Mel
`23
`None
`PD @6 wk
`700
`125
`890
`1800
`RCC
`19
`a Wk1D1, week 1 day 1; Wk1D4, week 1 day 4; Wk4D1, week 4 day 1; RCC, renal cell cancer; Mel, melanoma; SD, stable disease; PD,
`progressive disease.
`
`ability to sustain IFN-g induction. As shown in Fig. 2A (middle),
`only small differences in the peak and plateau levels of IL-15
`after the first, second, and seventh rhIL-12 doses were evident in
`patients with the type-I IFN-g pattern. In contrast, there was a
`50 –70% drop in the peak and plateau IL-15 levels by week 4 in
`patients with the type-II and type-III IFN-g patterns (Fig. 2,
`B-C, middle), and no IL-15 could be detected in the plasma
`before the seventh rhIL-12 dose in these patients.
`In all of the patients tested, small amounts of IL-18 were
`detected in the plasma before starting rhIL-12 (Fig. 2, A-C,
`bottom), with higher levels present before the second and sev-
`enth rhIL-12 doses. However, plasma IL-18 levels after an
`rhIL-12 injection usually peaked later than IFN-g. In addition,
`the loss of IFN-g production after the seventh rhIL-12 dose
`among patients with the type-II and type-III IFN-g patterns
`occurred despite continued IL-18 production at levels compara-
`ble with those measured immediately before and after the first
`rhIL-12 dose.
`IL-10 Induction and rhIL-12 Pharmacokinetics. As
`shown in Fig. 3A, IL-10 was induced after the first dose of
`rhIL-12 in a similar manner in all of the patients, returning to
`undetectable levels before the next rhIL-12 dose. A stronger
`increase in IL-10 production was observed after the second dose
`in all of the patients, with the highest peak levels detected in
`patients with the type-II IFN-g pattern (Fig. 3A, middle). How-
`ever, IL-10 induction continued after the seventh dose in all of
`the patients, including those with the type-I IFN-g pattern (Fig.
`3A, top), as well as those with the type-III pattern exhibiting a
`significant suppression of IFN-g (Fig