Vol. 4, 1183-1191, May 1998
`
`Clinical Cancer Research 1183
`
`Phase I Trial of Subcutaneous Recombinant Human Interleukin-12
`in Patients with Advanced Renal Cell Carcinoma
`
`Robert J. Motzer,1 Ashok Rakhit,
`Lawrence H. Schwartz, Thomas Olencki,
`Thomas M. Malone, Kate Sandstrom,
`Rosemary Nadeau, Harsukh Parmar, and
`Ronald Bukowski
`Genitourinary Oncology Service, Division of Solid Tumor Oncology,
`Department of Medicine [R. J.M., T. M. M.] and Department of
`Medical Imaging [L. H. S.], Memorial Sloan-Kettering Cancer Center,
`New York, New York 10021; Hoffmann-La Roche, Inc., Nutley, New
`Jersey, and Welwyn, United Kingdom [A. R., R. N., H.P.]; and the
`Cleveland Clinic Foundation, Cleveland, Ohio 44195 [T. 0., K. S.,
`R.B.J
`
`ABSTRACT
`Patients with advanced renal cell carcinoma were
`treated in a Phase I trial with escalating doses of recombi(cid:173)
`nant human interleukin-12 (rHuIL-12) given on days 1, 8,
`and 15 of each 28-day cycle. Treatment in the initial dose
`scheme consisted of a fixed dose with dose levels of 0.1, 0.5,
`and 1.0 p.g/kg given to cohorts composed of three or six
`patients. On the basis of the toxicity profile, a second scheme
`(up-titration) was undertaken wherein rHuIL-12 was esca(cid:173)
`lated for each patient from week 1 to week 2, to a target dose
`given week 3 and thereafter; cohort target dose levels were
`0.5, 0.75, 1.0, 1.25, and 1.5 p.g/kg. Fifty-one patients were
`treated: 32 (63%) had prior cytokine therapy and 19 (37%)
`had received no prior systemic therapy. The maximum tol(cid:173)
`erated dose for the faxed dose scheme was 1.0 p.g/kg. Dose(cid:173)
`limiting toxicities included increase in transaminase concen(cid:173)
`tration, pulmonary toxicity, and leukopenia. The most
`severe toxicities occurred with the first injection and were
`milder upon further treatment. With the up-titration dose
`scheme, the maximum tolerated dose was reached at 1.5
`p.g/kg, and dose-limiting toxicity consisted of an increase in
`serum transaminase levels.
`At the maximum tolerated dose of 1.5 p.g/kg, serum
`IL-12 levels increased to a mean peak level of 706 pg/ml.
`Serum levels of IFN--y increased to a mean peak level of
`about 200 pg/ml at 24 h after the first maintenance dose of
`1.5 p.g/kg. The best responses were as follows: one patient
`had complete response, 34 patients were stable, 14 patients
`showed progression, and 1 patient was inevaluable.
`
`In conclusion, rHuIL-12 was relatively well tolerated
`when administered by s.c. injection. The recommended dose
`according to the up-titration schedule of rHuIL-12 (p.g/kg)
`for Phase II trials was as follows: cycle 1, 0.1 (day 1), 0.5
`(day 8), 1.25 (day 15); cycle 2 onwards, 1.25. Phase II trials
`of rHuIL-12 were initiated in previously untreated patients
`with renal cell carcinoma and in patients with melanoma.
`
`INTRODUCTION
`rHulL-12 2 is a heterodimeric protein composed of two
`disulfide-linked subunits having molecular masses of 40 and 35
`kDa, respectively (I, 2). IL-12 as a cytokine has been shown to
`exert a number of regulatory effects on T lymphocytes and NK
`cells (3, 4). These include (a) enhancing the lytic activity of
`NK/LAK-cells; (b) facilitating specific cytolytic T-lymphocyte
`responses; (c) inducing the secretion of IFN--y by both T and NK
`cells; and (d) promoting the development of TH I-type helper T
`cells, thereby contributing to the development of cell-mediated
`immune responses (5-8). Other cell types, including mono(cid:173)
`cytes, macrophages, activated B-cells, dendritic cells, and kera(cid:173)
`tinocytes, also contribute to the biological activity of IL-12
`(5-8). One activity of IL-12 that may contribute to an antitumor
`effect is the ability to inhibit angiogenesis, an effect mediated
`through the induction of IFN--y (9).
`Interest in biological response modifiers as a treatment for
`metastatic renal cell carcinoma is fostered by the reproducible,
`albeit infrequent, responses with IFN-a and IL-2 against this
`chemotherapy-refractory malignancy ( 10). The antitumor activ(cid:173)
`ities of rHuIL-2 were demonstrated in a large number of murine
`tumor models, including Renea, a spontaneously arising renal
`cell carcinoma (8, 11 ). Treatment of mice bearing murine Renea
`renal cell carcinoma resulted in tumor growth inhibition, tumor
`regression, and prolongation of survival; the antitumor effect
`was superior to IL-2 and IFN-a (8, 11 ). These data provided the
`rationale for a Phase I trial of rHuIL-12 in patients with renal
`cell carcinoma. The schedule of s.c. administration at escalating
`dose levels of rHuIL-12 given once weekly for 3 weeks was
`based on single- and multiple-dose toxicology studies per(cid:173)
`formed in cynomolgus monkeys and chimpanzees. 3 Pharmaco(cid:173)
`kinetic and pharmacodynamic parameters were studied, which
`included assessment of IFN--y and serum neopterin, an uncon(cid:173)
`jugated pteridine released by macrophages after activation by
`IFN--y (12).
`
`Received 11/25/97; accepted 2/20/98.
`The costs of publication of this article were defrayed in part by the
`payment of page charges. This article must therefore be hereby marked
`advenisemenr in accordance with 18 U.S.C. Section 1734 solely to
`indicate this fact.
`1 To whom requests for reprints should be addressed, at Memorial
`Sloan-Kettering Cancer Center, 1275 York Avenue, New York.
`NY 10021.
`
`2 The abbreviations used are: rHuIL-12, recombinant human interleu(cid:173)
`kin-12; IL, interleukin; ASAT, aspartate aminotransferase; ALAT, ala(cid:173)
`nine aminotransferase.
`3 Roche, unpublished data.
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`1184 IL-12 in Renal Cell Carcinoma
`
`Table 1 Dosing schemes of rHuIL-12 (µ.g/kg)
`
`Cycle I
`
`Day 8
`Day I
`Cohort
`Dose escalation scheme A (fixed dose)
`I
`0.1
`0.1
`II
`0.5
`0.5
`III
`1.0
`1.0
`Dose escalation scheme B (2-step up-titration)
`I
`0.1
`0.25
`II
`0.1
`0.5
`III
`0.1
`0.5
`IV
`0.1
`0.5
`V
`0.1
`0.5
`
`Day 15
`
`0.1
`0.5
`1.0
`
`0.5
`0.75
`1.0
`1.25
`1.5
`
`Cycle 2 and
`others, days
`I, 8, and 15
`
`No. of
`patients
`
`Median no. of
`cycles (range)
`
`0.1
`0.5
`1.0
`
`0.5
`0.75
`1.0
`1.25
`1.5
`
`3
`15
`6
`
`3
`3
`3
`6
`12
`
`4 (1-9)
`4 (1-17)
`4 (.3-7)
`
`6 (2-16)
`2 (2-3)
`3 (2-4)
`4.5 (2-8)
`3 (2-11)
`
`PATIENTS AND METHODS
`Patients. Between April 1995 and November 1996, 51
`patients with advanced renal cell carcinoma were entered in this
`institutional review board-approved Phase I trial. All patients
`were between 18 and 75 years of age; gave informed consent;
`had measurable disease; had a Kamofsky performance status
`2:80%; had an estimated life expectancy of >4 months; had a
`WBC count of 2:3000 cells/mm3 , a granulocyte count of 2:2000
`cells/mm3, a platelet count of 2:75,000 cells/mm 3, and a hemo(cid:173)
`globin level of 2: 10 g/dl; had normal serum bilirubin and
`transaminase levels and alkaline phosphatase :s 2.5 times nor(cid:173)
`mal; had a normal serum creatinine concentration in patients
`without a prior nephrectomy and :s 1.5 times normal in patients
`with prior nephrectomy; and had a serum calcium level of
`:sl2.5 mg/di (:S3.12 mmol/liter). Exclusion criteria included
`active brain metastases, history of psychiatric disabilities or
`seizures, clinically significant cardiac abnormalities, chronic
`obstructive pulmonary disease, prior history of systemic liver
`disease, active systemic infection, and THI-mediated autoim(cid:173)
`mune diseases. Prior systemic therapy was allowed, but patients
`could not have received more than one previous chemotherapy
`plus one previous immunotherapy.
`rHuIL-12.
`rHuIL-12 was supplied by Hoffmann-La
`Roche, Inc. (Nutley, NJ) as ready-to-use HSA-free solution in
`single-dose glass vials containing 10, 100, 500, or 1000 µg of
`purified rHuIL-12 in 1 ml of sterile solution containing poly(cid:173)
`sorbate 80 (0.2 mg/ml) and 67 mM PBS adjusted to pH 7.0. The
`vials were stored at 2-8°C and protected from light. rHuIL-12
`was administered by s.c. injection using a 25 gauge needle.
`Dose Schedule. All patients were treated with two cycles
`of therapy lasting 28 days (Table 1 ). Each cycle consisted of s.c.
`injections on days 1, 8, and 15. Treatment was given on an
`outpatient basis, except that patients were hospitalized for phar(cid:173)
`macokinetic studies. On the basis of results of the tumor assess(cid:173)
`ment following cycle 2, patients with a response of stable
`disease or better received additional cycles of therapy until
`evidence of progression or unacceptable toxicity.
`In the initial dose scheme (scheme A), patients were treated
`with a fixed dose of rHuIL-12 at planned dose levels ofO. l, 0.5,
`and 1.0 µg/kg. Cohorts included three patients per dose level
`until a grade 2 or higher toxicity occurred, with the exception of
`grade 2 fever or Ieukopenia and grade 3 or 4 lymphopenia or
`fever; for these levels and subsequent dose-escalated levels, an
`
`additional three patients were entered. The maximum tolerated
`dose was defined as the level at which two of six patients
`experienced dose-limiting grade 3 or 4 toxicity.
`As a result of observations made during the initial phase of
`the trial regarding treatment tolerability, a second scheme
`(scheme B) was initiated in August 1995, after patients were
`treated on the trial. In this scheme, the dose of rHuIL-12 was
`escalated for each patient on days 8 and 15 of cycle 1 (Table 1).
`Subsequent cycles were administered at the day 15 dose level.
`Target (day 15) dose levels were 0.5, 0.75, 1.0, 1.25, and 1.5
`µg/kg. Patients weighing more than 80 kg received a maximum
`total dose corresponding to a body mass of 80 kg. The number
`of patients treated per dose level and the maximum tolerated
`dose was defined as described for dose scheme A.
`Patients were monitored by physical examination, com(cid:173)
`plete blood count, and serum chemistry on each day of treat(cid:173)
`ment. Each patient had a reassessment of measurable disease
`after the second cycle of treatment and then every 4 weeks
`thereafter. Patients with stable disease had tumor assessments
`performed every 2 months thereafter. All patients kept a daily
`log documenting symptoms and medications taken. Response
`was assessed according to WHO criteria and toxicity according
`to Common Toxicity criteria.
`Pharmacodynamic and Pharmacokinetic Parameters.
`IFN--y, tumor necrosis factor-a, and neopterin concentrations
`were measured in the serum of patients at preselected times.
`Serum samples were obtained at baseline and 10, 24, 48, 72, 96,
`and 168 h following treatment on day 1 of cycle 1 of treatment
`scheme A and day 15 of cycle 1 of treatment scheme B.
`IFN--y concentrations in serum were determined by a com(cid:173)
`mercial assay (R&D Systems, Minneapolis, MN). In brief, 50 µl
`of the assay diluent were added to each well of the microtiter
`plate. Two hundred µ1 of standard or sample was added to each
`well and incubated for 2.5 h at room temperature. Each well was
`then washed three times with buffer, and any remaining wash
`removed by blotting it against a clean paper towel or by aspi(cid:173)
`ration. Two hundred µl of IFN--y conjugate were added and
`incubated for 2 hat room temperature. Each well was repeatedly
`washed/aspirated, 200 µl of substrate solution were added, and
`the samples were incubated for 20 min. Fifty µl of stop solution
`were added to each well and tapped to ensure thorough mixing.
`The absorbance of each well was determined within 30 min
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`

`Clinical Cancer Research 1185
`
`using a spectrophotometer set at 450 nm. The interassay varia(cid:173)
`bility was < 10%, and the limit of detection was 3.0 pg/ml.
`Neopterin concentration in serum was measured using a
`radio-immunoassay that used 1251-labeled neopterin as a tracer
`(lncstar, Stillwater, MN). Antineopterin rabbit antibody was
`incubated with samples, standards, and 1251-labeled neopterin.
`After a 1-h incubation at 37°C, antirabbit antiserum from sheep
`in polyethylene glycol buffer was added and incubated at room
`temperature for 15 min. Samples were centrifuged, supernatant
`was decanted, and pellets were counted for radioactivity. The
`amount of neopterin in sample was inversely proportional to the
`amount of radioactivity in the pellet. Unknown concentrations
`were calculated from a standard curve. The assay showed neg(cid:173)
`ligible cross-reactivity with other neopterin-like compounds
`(biopterin, monapterin, and tetrahydroneopterin) and a lower
`limit of quantitation of 0.2 ng/ml.
`Tumor necrosis factor-a concentrations were determined
`by a quantitative sandwich enzyme-immunoassay technique
`(R&D Systems). Tumor necrosis factor-a standards and study
`samples were incubated with antibody bound to a microtiter
`plate. After washing away unbound substances, an enzyme(cid:173)
`linked polyclonal antibody specific for tumor necrosis factor-a
`was added to the wells. The plates were washed to remove
`unbound antibody-enzyme reagent, and a substrate solution was
`added. Color development was proportional to the amount of
`tumor necrosis factor-a bound in the initial step. Sample con(cid:173)
`centrations were determined on a standard curve by plotting the
`absorbance versus tumor necrosis factor-a concentration. The
`lower limit of quantitation of the assay was 15.6 pg/ml.
`Serum concentrations of rHuIL-12 were measured at base(cid:173)
`line and at 2, 4, 8, IO, 16, 24, 30, 34, 48, and 72 hon days 1-3
`of cycle I of scheme A (fixed dose) and on day 15 of cycle I
`when first maintenance (target) dose was administered in
`scheme B (up-titration schedule). rHuIL-12 concentration was
`measured by a two-step method of antibody capture to ensure
`specificity followed by a cell proliferation assay. rHuIL-12 was
`isolated from serum by an affinity technique that involved
`incubation of samples in sterile tissue culture plates precoated
`with mouse antihuman IL-12 monoclonal antibody (13).
`Samples and rHuIL-12 standards were incubated with the
`bound monoclonal antibody for 3 h on an orbital shaker at room
`temperature. After a sterile wash with PBS to remove all non(cid:173)
`specific material, KIT 5/K6 cells were added to each well of the
`tissue culture plates. After incubation for 66 h, cells were pulsed
`with [methyl-3H]thymidine for six h, and cell proliferation was
`measured by [methyl-3H]thymidine incorporation. Sample val(cid:173)
`ues were determined on a standard curve obtained from plotting
`radioactive counts against IL-12 concentration. The assay had a
`lower limit of detection of 50 pg/ml of serum using 100-µl
`aliquots. The interassay precision was 8.0%. rHulL-12 is stable
`in serum for 24 h at room temperature. Serum samples were also
`found to be stable after three freeze/thaw cycles.
`Anti-rHuIL-12 antibodies were measured in serum at base(cid:173)
`line and periodically thereafter. Anti-rHuIL-12 antibodies were
`determined by a sandwich enzyme immunological assay. The
`assay was based on the ability of the multivalent anti-IL-12
`antibodies to simultaneously bind rHuIL-12 coated on the wells
`of microtiter plate and soluble peroxidase-conjugated rHuIL-12.
`The intra- and interassay variabilities were 11 and 20%, respec-
`
`Table 2 Patient characteristics
`
`Characteristic
`
`Patients
`Male/female
`Median age (range)
`Median Kamofsky performance status
`(range)
`Prior nephrectomy
`Yes
`No
`Prior therapy
`IFN
`IL-2
`IFN plus IL-2 with/without 5-
`fluorouracil or floxuridine
`Granulocyte-macrophage colony-
`stimulating factor plus IL-6
`Chemotherapy (Tallamustine)
`Hormonal therapy (Tamoxifen)
`Radiotherapy
`No systemic therapy
`Evaluable sites
`Lung
`Mediastinum
`Retroperitoneal lymph node
`Kidney
`Adrenal gland
`Bone
`Liver
`Spleen
`Peripheral node
`Muscle
`Skin
`
`No.(%)
`
`51
`38 (74)/13 (26)
`56 (38-73)
`90 (80-90)
`
`43 (84)
`8 (16)
`
`14 (27)
`6 (12)
`11 (26)
`
`1 (2)
`
`2 (4)
`1 (2)
`6 (12)
`19(37)°
`
`31 (61)
`9 (18)
`9 (18)
`5 (10)
`5(10)
`5 (10)
`5 (10)
`2(4)
`2(4)
`I (2)
`I (2)
`
`"Includes two patients treated with radiation therapy.
`
`tively. The level of detection for this assay was 29 ng/ml. The
`specificity of the assay for anti-IL-12 antibodies was confirmed
`by the absence of any measured response in serum samples
`containing antibodies against various irrelevant proteins.
`
`RESULTS
`Patient Characteristics. Fifty-one patients were treated
`with rHuIL-12 (Table 2). The median age was 56 years, and 43
`(75%) had a prior nephrectomy. Thirty-two (63%) had prior
`cytokine therapy (IFN-a, IL-2, or IL-6), and 19 (37%) had
`received no prior systemic therapy.
`Treatment Administered and Toxicity. Twenty-four
`patients were treated with a fixed-dose schedule (scheme A) and
`27 were treated with the up-titration schedule (scheme B; Table
`I). Fever, fatigue, and a rapid, transient decrease in WBC counts
`after the first injection were common to all dose levels. The
`decrease in WBC count included a decrease in lymphocytes and
`neutrophils; the lymphocyte count decreased to a greater extent.
`Other frequent adverse events at doses equal to or greater than
`0.5 µg/kg were mild to moderate chills, diaphoresis, anorexia,
`headaches, transient cough, and nausea and vomiting. There
`were no treatment-related deaths, and none of the patients re(cid:173)
`quired intensive care unit support.
`With the fixed-dose regimen, the 0.1 µg/kg dose was well
`tolerated, and there were no grade 3 toxicities (except fever,
`which was not dose-limiting). One of three patients treated with
`0.1 µg/kg rHuIL-12 had a grade 2 increase in serum transami-
`
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`

`1186 IL-12 in Renal Cell Carcinoma
`
`Table 3 Number of patients experiencing selected adverse events in cycle I, day 1, versus cycle 2, day 15 (grades 2, 3, or 4), from fixed dosing
`scheme A
`
`0.1 µ.g/kg (n = 3)
`
`0.5 µ.g/kg (n = 15)
`
`1.0 µ.g/kg (n = 6)
`
`Adverse event
`Fever
`WBC
`ASAT
`ALAT
`
`Cycle I,
`day I
`(2,0)
`(0, 0)
`(1. 0)
`(1,0)
`
`Cycle 2,
`day 15
`(0, 0)
`(0, 0)
`(0, 0)
`(0, 0)
`
`Cycle I,
`day 1
`(8. 2)
`(3, 1)
`(0, 0)
`(1,0)
`
`Cycle 2,
`day 15
`(1,0)
`(1. 0)
`(0,0)
`(0, 0)
`
`Cycle I,
`day 1
`(4,0)
`(2, 1)
`(1. 1)
`(0, 1)
`
`Cycle 2.
`day 15
`(1,0)
`(0,0)
`(1. 0)
`(0, 0)
`
`4IO r-------------------------------------
`
`-3IO
`
`180
`
`100
`
`· · · · · · ·
`
`· · · -&rade~ · · · · · · · · · · · · · · · · · · · · ·
`
`IO
`
`..... ·Grac1e1· .......................................•.................................
`
`Grade
`a
`u u
`
`C
`
`Q
`u
`
`0
`
`ID
`
`I
`
`"' I N I .,. !
`
`Q
`u
`
`Q
`~
`
`u
`Q
`u
`Fig. I Changes in ALAT levels following fixed doses of weekly s.c. administration of rHuIL-12 in a patient (patient 103) at 1.0 µ.g/kg dose.
`Increases in transaminase levels were highest after the first dose and decreased on subsequent dosing.
`
`nase concentration in cycle I, but the patient met grade I
`toxicity pretreatment.
`Fifteen patients were treated with a fixed-dose schedule of
`0.5 µg/kg. All reported a mild to moderate fever within 36 h of
`the first injection, and all had mild to moderate increases in
`serum transaminase concentrations after the first injection. One
`patient had grade 4 gastrointestinal toxicity. This patient had a
`distant history of recurrent colitis and a family history of ulcer(cid:173)
`ative colitis undisclosed at study entry. He developed bloody
`diarrhea during the second cycle, and a colonoscopy showed
`pancolitis. The patient was taken off the study and improved
`with medical management that included steroids.
`Six patients were treated with a fixed-dose schedule of 1.0
`µg/kg; two experienced dose-limiting toxicity composed of a
`grade 3 increase in transaminase concentration plus grade 4
`pulmonary toxicity in one patient and a grade 3 leukopenia in
`
`one patient. The pulmonary toxicity was characterized by acute
`onset of shortness of breath in the setting of fever approximately
`40 h following day I of cycle I treatment during hospital stay.
`With supplemental oxygen, the patient recovered immediately
`without sequelae. Evaluation included a chest radiograph, elec(cid:173)
`trocardiogram, and ventilation/perfusion scan, which failed to
`reveal an etiology. The patient weighed 112 kg, and the rela(cid:173)
`tively high dose of 112 µg of rHuIL-12 may have been a factor;
`subsequent patients treated on the study were dosed according to
`a maximum 80 kg weight. At the 1.0 µg/kg dose treatment,
`fever and leukopenia were more persistent with slower recovery.
`Nearly all patients treated with 1.0 µg/kg had an increase in
`transaminase levels 5-10 days after the first injection, but only
`one patient had grade 3 toxicity.
`A comparison of the severity of fever, leukopenia, and
`elevated serum transaminase concentration according to dose in
`
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`

`Clinical Cancer Research 1187
`
`Table4 Number of patients experiencing selected adverse events in cycle 1 versus cycle 2 (grades 2, 3, or 4) from up-titration dosing scheme B
`0.5 µ.g/kg (n = 3)
`0.75 µ.g/kg (n = 3)
`1.0 µ.g/kg (n = 3)
`1.25 µ.g/kg (n = 6)
`1.5 µ.g/kg (n = 12)
`
`Adverse event
`Fever
`WBC
`ASAT
`ALAT
`
`Cycle 1,
`day 15
`(0,0)
`(0, 0)
`(0, 0)
`(I, 0)
`
`Cycle 2,
`day 15
`(0, 0)
`(1,0)
`(0, 0)
`(0, 0)
`
`Cycle I,
`day 15
`(I, 0)
`(I, 0)
`(0,0)
`(I, 0)
`
`Cycle 2,
`day 15
`(0, 0)
`(1,0)
`(0, 0)
`(0, 0)
`
`Cycle I,
`day 15
`(2, 0)
`(2, 0)
`(0, 0)
`(0, 0)
`
`Cycle 2.
`day 15
`(I. 0)
`(1,0)
`(I, 0)
`(0, 0)
`
`Cycle 1,
`day 15
`(2,0)
`(2, 0)
`(0,0)
`(0,0)
`
`Cycle 2.
`day 15
`(I. 0)
`(3, 0)
`(0,0)
`(0,0)
`
`Cycle I.
`day 15
`(3,0)
`(4,0)
`(0, 1)
`(0, I)
`
`Cycle 2,
`day 15
`(0,0)
`(4, 0)
`(0, I)
`(I, 1)
`
`100 0 . - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -
`
`900
`
`800
`
`700
`
`300
`
`200
`
`100
`
`..............
`..
`..
`...
`0 .,
`.,
`.,
`.,
`.,
`.,
`.,
`.,
`....
`.,
`.,
`.,
`"'
`N
`"'
`.5
`C
`0
`Ei
`Ei
`~ C
`Ei
`Ei
`ai u u C u
`0
`u e ~ ~ e
`0
`c5
`u u ..... u u u u
`.....
`C
`~
`"'
`ca
`a,
`"'
`Ei
`Ei
`..,
`Ill
`C
`Ei
`C
`C
`Ei
`u
`c5
`c5
`l3
`u
`u
`l3
`Fig. 2 Changes in ALA T levels following slow intrapatient dose escalation of rHuIL-12 in a patient (patient 120) at a 1.5 µ.g/kg maintenance dose.
`Grade 3 increases occurred after 4 weeks of treatment and decreased on subsequent dosing at 50% level.
`
`0,
`C
`N
`
`.,
`"'
`0
`C
`.....
`a,
`C
`N
`u
`
`0
`
`0
`
`0
`
`0
`
`N
`C
`
`0,
`C
`
`0
`
`N
`
`0,
`
`N
`N
`C
`
`N
`C
`N
`u
`
`.,
`.,
`0
`C
`.....
`Ei
`N
`u
`
`cycle 1 versus cycle 2 suggested a dose-effect relationship
`(Table 3). At all dose levels, the most severe toxicities occurred
`mainly after the first injection and were milder upon further
`treatment with rHuIL-12 (Fig. 1). Most patients showed only
`mild (grade 1) fever and other common adverse events (leuko(cid:173)
`penia and elevated ALA T and/or ASA T) in cycle 2 at all doses.
`Therefore, the maximum tolerated dose was reached at 1.0
`µg/kg with the first dose. Because tolerability improved with
`subsequent therapy, a slow escalation dosing scheme was fol(cid:173)
`lowed for a second cohort of patients.
`In the second dose scheme, the dose of rHuIL-12 was
`up-titrated in two steps for each patient following day 1 and day
`8 treatments, and patients were treated with the maintenance
`dose ranging from 0.5 to 1.5 µg/kg from day 15 of cycle 1 until
`they were taken off study or required a dose reduction. No
`dose-limiting toxicity was observed in patients treated at target
`maintenance dose levels of 1.25 µg/kg or less. The maximum
`
`tolerated dose was 1.5 µg/kg. At this dose level, two of the first
`six patients had dose-limiting toxicity. The one patient had
`grade 4 serum transaminase (ALAT) concentrations during
`week 2 of cycle 2 at the maintenance dose of 1.5 µg/kg. A
`second patient had grade 3 serum transaminase elevations fol(cid:173)
`lowing the first injection of the 1.5 µg/kg dose during cycle 1.
`An additional six patients were treated at this dose level without
`any dose-limiting toxicity.
`The severity of changes in serum transaminase and leuko(cid:173)
`cyte concentrations, as well as fever, was compared between
`cycle 1 and cycle 2 following escalation doses (Table 4 ). In
`contrast to patients treated with the fixed dosing scheme, the
`slow escalation of rHuIL-12 was better tolerated, although sim(cid:173)
`ilar adverse events occurred at later times ( cycle 2) and at higher
`doses (Fig. 2).
`An additional toxicity observed, albeit not dose-limiting,
`was stomatitis. Grade I mucositis was first noted in two patients
`
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`
`
`Research.
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`Breckenridge v. Novartis, IPR 2017-01592
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`

`

`1188 IL-12 in Renal Cell Carcinoma
`
`1000
`
`900
`
`800
`
`700
`
`--+-1.0 ug/kg; Fixed C1D1
`--0.5 ug/kg; Esc C1D15
`--1.0 ug/kg; EscC1D15
`--M--1.25 ug/kg; EscC1D15
`---1.50 ug/kg; Esc C1D15
`
`C, 600
`E
`~
`8 500
`_,
`:i t
`
`N
`
`400
`
`Fig. 3 Mean serum concentration pro(cid:173)
`files of rHuIL-12 after the first dose of
`1.0 µ.g/kg in the fixed dose scheme com(cid:173)
`pared to the first maintenance dose of
`0.5-1.5 µ.g/kg in the intrapatient escala(cid:173)
`tion dose scheme.
`
`300
`
`200
`
`100
`
`0
`
`0
`
`2
`
`4
`
`8
`
`10
`
`16
`Time (hr)
`
`24
`
`30
`
`34
`
`48
`
`Table 5 Mean (:!:SE) pharmacokinetic parameters of rHuIL-12 at various doses (cycle I, day 1, for fixed dose and cycle I, day 15, for
`escalation doses)
`
`Fixed Dose
`
`Escalation Dose
`
`0.5 µ.g/kg
`(n = 15)
`320 (70)
`13
`7031 (2031)
`13 (2)
`
`Cmax (pg/ml)
`T max (h)
`AUC" (pg-h/ml)
`l112 (h)
`"AUC, area under the curve.
`h Adequate sample in one patient.
`
`1.0 µ.g/kg
`(n = 6)
`1092 (275)
`16
`26589 (6633)
`10 (2)
`
`0.5 µ.g/kg
`(n = 3)
`70 (35)
`16
`454 (237)
`9b
`
`1.0 µ.g/kg
`(n = 3)
`353 (79)
`14
`7631 (2745)
`15 (7)
`
`1.25 µ.g/kg
`(n = 6)
`383 (53)
`15
`9109 (1705)
`16 (3)
`
`1.5 µ.g/kg
`(n = 12)
`706 (159)
`15
`14269 (3145)
`12 (I)
`
`treated at the 1.0 µg/kg dose level in the fixed-dose scheme and
`one patient treated at the 1.0 µg/kg dose level in the up-titration
`dose scheme. When higher doses of rHuIL-12 were given in the
`up-titration dose scheme, the severity increased and reached
`grade 2 toxicity in two patients treated at 1.25 µg/kg and two
`patients treated at 1.5 µg/kg.
`Phannacokinetics/Phannacodynamics. Serum concen(cid:173)
`trations of rHuIL-12 are shown in Fig. 3 at various doses after
`the first dose (day l) in fixed dose scheme A or after the first
`maintenance dose (day 15) in escalation dose scheme B. Serum
`concentration of IL-12 increased slowly after s.c. administra(cid:173)
`tion, with peak serum concentration observed between 8 and
`24 h. Serum concentration then decreased gradually with a
`half-life ranging from 7 to 21 h. Mean pharmacokinetic param(cid:173)
`eters of IL-12 are described in Table 5 for both dose schemes.
`The serum concentrations of IL-12 for equivalent target doses
`were lower after escalation dose scheme B compared to fixed
`
`dose scheme A, although the peak time and half-life were
`unchanged.
`After treatment for as long as 8 months, no serum antibod(cid:173)
`ies were identified against rHuIL-12. Serum levels of anti(cid:173)
`rHulL-12 antibody were below the detection limit (29 ng/ml) in
`all patients.
`Three different immunological markers (IFN--y, neopterin,
`and tumor necrosis factor-a) were followed to investigate THI
`immune stimulation by rHuIL-12. Serum levels of IFN--y were
`nonmeasurable (<3.0 pg/ml) at baseline. The mean peak level
`achieved in patients treated with 1.0 µg/kg by the fixed dose
`schedule was 250 pg/ml (Fig. 4). Similar to the levels of serum
`IL-12, IFN--y increased but to a lesser extent when dosing
`scheme was changed from fixed to the up-titration schedule. The
`peak level of IFN--y was 126 pg/ml at 1.0 µg/kg dose in the
`escalation dose scheme. The levels, however, increased as the
`dose was increased within the escalation scheme to 205 pg/ml at
`
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`Page 6 of 10
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`

`

`Clinical Cancer Research 1189
`
`250 ...... - - - - - - - - - - - - - - - - - - - - - - - - - - - .
`
`--1.0 ugkg; Fixed C101
`...... o.5 ug.'llg; Eec: C1015
`-+-1.0 ug.'llg; Eec: C1D15
`--125 ug.'llg; Eec: C1015
`-+-1.50 ug.'llg; Eec: C1D15
`
`Fig. 4 Mean serum concentration profiles
`of IFN--y after the first dose of rHuIL-12 at
`1.0 µ.g/kg in the fixed dose scheme com(cid:173)
`pared to the first maintenance dose of
`rHuIL-12 at 0.5-1.5 µ.g/kg in the intrapa(cid:173)
`tient escalation dose scheme.
`
`200
`
`50
`
`0F-----.,:::.----+------+------+--------1
`72
`0
`24
`48
`10
`98
`
`Time lhr)
`
`--1.0 ugkg; Fixed C101
`...... o.5 ug.'llg; Elc C1D15
`-+-1.0 ug/kg; Elc C1015
`--1.25 ug/kg; Elc C1015
`-+-1.50 ug/kg; Elc C1D15
`
`Fig. 5 Mean serum concentration profiles of
`neopterin after the first dose of rHuIL-12 at
`1.0 µ.g/kg in the fixed dose scheme compared
`to first maintenance dose of rHuIL-12 at 0.5-
`1.5 µ.g/kg in the intrapatient escalation dose
`scheme.
`
`IO
`
`50
`
`20
`
`o----------+---------+----------1
`0
`10
`24
`48
`111
`72
`98
`Tlmelhr)
`
`1.5 µg/kg dose, demonstrating a dose-related increase in T8 1
`stimulation. The onset of such stimulation was relatively rapid,
`with peak time for IFN--y occurring at about 24 h after IL-12
`administration. The levels then decreased during subsequent
`days of the cycle to nonmeasurable levels by day 8 of next dose.
`
`Neopterin serum concentration increased more slowly than
`IFN--y, with peak concentration reached between 72 and 96 h
`following treatment with rHuIL-12. The predose baseline neop(cid:173)
`terin concentration ranged from 1.2 to 8 ng/ml. The mean ± SE
`peak neopterin concentration after the first dose of 1.0 µg/kg
`
`Downloaded from
`
`on February 22, 2018. © 1998 American Association for Cancerclincancerres.aacrjournals.org
`
`
`Research.
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`NOVARTIS EXHIBIT 2039
`Breckenridge v. Novartis, IPR 2017-01592
`Page 7 of 10
`
`

`

`1190 IL-12 in Renal Cell Carcinoma
`
`was 70 ± 25 ng/ml. For patients treated with the up-titration
`schedule (scheme B), the mean ± SE peak neopterin levels after
`the first maintenance (week 3) dose were 15.4 ± 2.7, 19.5 ±
`7.9, 35.0 ± 11.2, and 31.0 ± 5.0 ng/ml at 0.5, 1.0, 1.25, and 1.5
`µg/kg doses, respectively (Fig. 5). The peak concentration oc(cid:173)
`curred at about 96 h and maintained at higher than predose
`levels prior to administration of the next dose (168 h). Few
`sporadic levels of tumor necrosis factor-a were detected during
`the treatment, but no consistent increase in levels was observed
`in relation to severity of the measured adverse events.
`Response and Survival. Fifty patients were evaluable
`for response. One patient, who was considered inevaluable,
`received day I of treatment and was removed from study be(cid:173)
`cause of pulmonary toxicity. Two patients discontinued treat(cid:173)
`ment because of progression following one month of therapy. At
`the 2-month tumor assessment following two cycles of therapy,
`the response was as follows: stable, 34 patients; progressive, 14
`patients. One patient with a response of stable disease following
`the first two cycles had tumor regression and subsequently
`achieved a complete response. The extent of disease in this
`patient was confined to lung parenchyma, and he was treated
`with 1.5 µg/kg of rHuIL-12 according to the up-titration sched(cid:173)
`ule. This patient continues on treatment following 12 cycles of
`therapy and remains progression-free at > 12 months. For the
`remaining 34 patients with a best response of stable, the median
`time to progression was 4 months (range, 2-15 months).
`
`DISCUSSION
`Common adverse events observed in this study were fever,
`chills, fatigue, transient leukopenia, and increase in serum con(cid:173)
`centrations of hepatic transaminase. Other toxicities included
`stomatitis, respiratory distress in the setting of fever, and colitis.
`The colitis may have represented reactivation of an autoimmune
`disorder; rHuIL-12 facilitated the development of autoimmune
`disease in several murine models (