(CANCER RESEARCH 49. 6521-6528. December I. 1989|

Schedule Dependency of the Antitumor Activity and Toxicity of PolyethyleneGlycol-modified Interleukin 2 in Murine Tumor Models

Robert J. Zimmerman,1 Sharon Lea Aukerman, Nandini V. Katre, Jeffrey L. Winkelhake, and John D. Young

Department of Pharmacology [R. J. Z.. S. L. A.. J. L. H., J. I). }'./ and Department of Process and Product Development [N. K K.J, Cetus Corporation.

Emeryville, California 9461)8

ABSTRACT

Modification of recombinant human intcrlcukin 2 (rhIL-2) with mon-omcthoxy polyethylene glycol has been shown to alter its pharmacoki-

netic properties. Therefore, we investigated the pharmacological parameters of schedule and dose in order to assess the impact on the in viroantitumor activity of this modification. The antitumor efficacy, as well asthe toxicity, of polyethylene glycol-interleukin 2 (PEG-IL-2) was compared to that of rhIL-2 in three transplantable syngeneic murine tumormodels. Molli A fibrosarcoma, B16 melanoma, and Pan-02 pancreaticcarcinoma. At cquitoxic dose levels, the antitumor activity of PEG-IL-2was far superior to that of rhIL-2 in all three tumor models. This efficacyof PEG-IL-2 was dose dependent and was greatest on a Q7D x 2 schedule

in Meth A and B16. When the same total doses were further divided anddelivered on any of several alternative schedules, either the efficacy wasreduced or the toxicity of the treatments was increased. In Pan-02, arhIL-2-resistant tumor, PEG-IL-2 treatment on either the Q7D x 2,

Q4D x 3, or Q3D x 4 schedule resulted in approximately a 200%increase in lifespan; however, the toxicity of the treatment increased asthe interval between doses was shortened. Simulations of the pharmaco-

kinetic profiles of these various regimens suggested that the toxicity ofPEG-IL-2 and rhIL-2 was related to the minimum plasma concentration

that was obtained and the time interval between peak levels. The efficacyof the treatment was associated with the intcrleukin 2 plasma peakheight, since a dose response was observed; however, peak plasmaconcentration did not appear to be the only parameter which determinedefficacy. We hypothesize that this observed schedule dependence is alsoaffected by the kinetics of the host's biological response to rhIL-2.

INTRODUCTIONThe therapeutic activity of rhIL-22 has been investigated in

preclinical murine tumor models by a number of investigators(Refs. 3-12 and references therein). The clinical activity of thislymphokine has been demonstrated both as a single agent andin combination with lymphokine-activated killer cells (Refs.13-19 and references therein). Modification of rhIL-2 withmonomethoxy PEG produces a biologically active rhIL-2 with

enhanced solubility and an altered pharmacokinetic profile (1).The in vivo activity of a Mr 95,000 PEG-IL-2 species administered daily i.p. was investigated in the Meth A model and wasshown to be enhanced in terms of both efficacy and toxicity (2).

In order to more thoroughly study the influence of PEGmodification on the bioactivity of rhIL-2, a series of PEG-IL-2species have been produced and the in vivo biological activityof some of these compounds has been characterized. The degreeof modification affects the plasma half-life of PEG-IL-2 (1);therefore, we hypothesized that these changes in clearance

Received 3/23/89; revised 8/1/89: accepted 8/24/89.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 inaccordance with 18 II.S.C. Section 1734 solely to indicate this fact.

1To whom requests for reprints should be addressed, at Department ofPharmacology, Cetus Corporation, 1400 Fifty-third Street, Emeryville, CA94608.

2The abbreviations used are: rhIL-2, recombinant human interleukin 2: PEG-IL-2. monomethoxy polyethylene glycol-modified recombinant human interleu-kin 2: QD. every day: Q7D x 2. every 7 days. 2 times: Q4D x 3. every 4 days. 3times: Q3D x 4, every 3 days. 4 times; Q2D x 5, every 2 days. 5 times; PEG.polyethylene glycol: BDF,. C57BL/6J X DBA/2J F,: IL-2. interleukin 2.

would also have an impact on the choice of the in vivo treatmentschedule which would result in the greatest efficacy and leasttoxicity. The present series of studies was conducted using rhlL-2 modified to an average of 2 to 3 molecules of M, 7,000 PEG/rhIL-2 molecule, which produced a PEG-IL-2 species with anapparent average molecular weight of about 160,000 by sizeexclusion chromatography. The pharmacokinetic data suggested that the optimal treatment schedule for PEG-IL-2 wouldbe one with less frequent administration than the schedule forrhIL-2, for example once a week compared to daily dosing. Thetumor model data showed that the efficacy obtainable withPEG-IL-2 was superior to that of rhIL-2, and this improvementin efficacy was not associated with an increase in toxicity asjudged by lethality.

MATERIALS AND METHODS

Animals. Specific pathogen-free, 18-20 g, female BALB/c. C57BL/6. and BDF, mice were obtained from Charles River Laboratories(Wilmington, MA). Mice were weight matched, ear notched, and randomized at the beginning of each experiment. The details of our animalhusbandry conditions have been described (7).

Tumor Cell Lines. B16W10, a subline of B16 melanoma, was maintained and implanted s.c. as described (7). Briefly, cultured cells wereharvested by routine trypsinization techniques and washed by centrifu-gation, and 5 x IO6 viable cells were inoculated in the suprascapular

area of BDF, mice. The Meth A fibrosarcoma was the generous gift ofDr. Lloyd Old and was carried as an ascitic tumor stock in BALB/cmice. One million viable cells were inoculated s.c. in the suprascapulararea for efficacy experiments. Pan-02 was the generous gift of Dr. W.R. Leopold and was carried s.c. in C57BL/6 mice for tumor stocks.For experiments, a brei was prepared and 100 /jl were injected s.c. inthe suprascapular area. All cell lines and hosts were negative forMycoplasma and murine viral contamination (MAP test; Charles RiverProfessional Services).

Therapeutics. Highly purified rhIL-2 from Escherichia coli was storedas a lyophilized powder, reconstituted in sterile water for injection(LyphoMed). and further diluted as necessary in 5% dextrose in water(20). The endotoxin contamination was less than 0.1 EU/mg rhIL-2.PEG-IL-2 was produced and purified as previously described (1, 2),with a degree of modification equivalent to 2 to 3 PEG M, 7,000molecules/rh.IL-2 molecule. The apparent average molecular weight ofthis species was M, 160.000 by size exclusion chromatography, whichis a measure of the hydrodynamic radius of the molecules. The endotoxin content was 0.4 EU/mg rhIL-2. The specific activity of the PEG-IL-2 and rhIL-2 was 4 x 10*and 18.0 x IO6lU/mg respectively.

Antitumor Efficacy. Meth A and Pan-02 tumor cells were inoculateds.c. 7 days prior to the initiation of therapy. Therapy was initiated theday after s.c. injection of B16 tumor cells. Therapeutics were administered i.v. in 0.1-ml volume. In order to make meaningful comparisonsof the activities of rhIL-2 and PEG-IL-2, dose levels were based on theIL-2 protein concentration of the dosing solution. Tumor volumes werecalculated as the product of caliper measurements taken in threeorthogonal directions. Tumor-bearing hosts with apparently completeregressions at the s.c. site of inoculation in Meth A were followed for35 days, or in B16 for at least 60 days, to assess the durability of thisresponse. Since the Pan-02 tumor spontaneously metastasizes from thes.c. site of injection to regional lymph nodes and lung (21), the survivalof the hosts, as well as the size of the s.c. tumor mass, was used as an

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ENHANCED THERAPEUTIC INDEX OF l'EG-lL-2

indication of the therapeutic benefit of the treatments.Antitumor Kfficacy Data Analysis. Data are reported as the means of

replicate experiments using 5 or 6 mice/dose group/experiment. Insome combinations which produced high levels of lethality, particulardoses or schedules were not repeated (see tables for details). For MethA, the T/C% was determined as the mean tumor volume at day 21 ofthe treated group/mean tumor volume at day 21 of the control group.For B16, the tumor growth delay parameter T—C was defined as thenumber of days for the mean tumor size of the treated group to reach500 mm' subtracted from the number of days for the mean tumor sizeof the controls to reach 500 mm'. Survival data for Pan-02 are expressed

as the percentage increase in lifespan. calculated as the median numberof survival days of the treated group/median number of survival daysof the control group x 100. Statistical comparisons to controls oramong various experimental groups were performed using the Quaderank analysis of covariance; P was set at 0.05.

Pharmacokinetics. Male CD mice weighing 18 to 22 g were obtainedfrom Charles River Laboratories (Wilmington, MA). Five mice wereinjected/sample time with 0.25 mg PEG-IL-2/kg (total of 60 mice),and three mice were injected/sample time with 7.5 mg rhIL-2/kg (totalof 36 mice). The dose was injected into a lateral tail vein, and onesample of blood was obtained from each mouse by retroorbital sinuspuncture. Standards were prepared in mouse plasma and assayed withthe samples. Plasma was assayed for 1L-2 bioactivity by measuring theincorporation of pHjthymidine into DNA following incubation ofplasma in the presence of HT2 cells, mouse fibroblasts which aredependent on 1L-2 for growth (22, 23). Mean values of rhIL-2 bioactivity and PEG-IL-2 bioactivity adjusted for dose (x 30) were analyzed toobtain the macroconstants (coefficients and rate constants) which described the plasma curves, using the microcomputer program RSTRIPobtained from MicroMath Scientific Software (Salt Lake City, UT).Coefficients A, B, and C were adjusted for dose to simulate variousdosage schedules using the microcomputer program PCNONLIN (24),obtained from Statistical Consultants, Inc. (Lexington, KY). The rateconstants were assumed to be fixed for different doses and schedules.

RESULTS

Schedule Dependence in Meth A. The Meth A model wasinvestigated initially because it was known to be sensitive torhIL-2 under a variety of protocols, probably due to the tumor's

immunogenic nature (25). Table 1A presents the results ofdose-response studies in which rhIL-2 was given i.v. once dailyfor 9 days (QD x 9). The maximum dose administered was12.5 mg/kg/dose (112.5 mg/kg total dose) due to solubilitylimitations of the rhIL-2 and i.v. dosing volume restrictions.This dose level was near the maximum tolerated dose, however,as 1 of 10 hosts died due to treatment. Complete regressions atthe s.c. site of injection were obtained in 3 of 10 and 4 of 10 ofthe animals in the two highest dose groups. Further, there weremodest dose-dependent antitumor effects observed at the twolower dose levels, as shown by the T/C% (Table 1A).

In order to keep some parameters constant across the varioustumor models and to develop some basis for therapeutic benefitcomparisons, it was necessary to carefully choose the schedulesand doses for evaluation of PEG-IL-2. It was desirable (a) toobtain some toxicity, in order to make comparisons at equitoxicdose levels, which then addresses the issue of therapeutic index,and (b) to allow about the same time interval between the lastdose and the final tumor volume determination, in order toallow any therapeutic benefits to appear. Therefore, three doselevels were investigated and the intervals between repeat doseswere chosen to maintain approximately the same total treatment period.

As shown in Table IB, four different treatment scheduleswere used: Q7D x 2, Q4D x 3, Q3D x 4, and Q2D x 5. Thethree total dose levels of 50, 25, or 12.5 mg/kg PEG-IL-2 were

Table 1 rML-2 (A) and PEíi-IL-2(B) antitumor efficacy in Melh .1

Total rhIL-2 Number deaths/ CompleteA. dose (mg/kg)° 7"/C%* total treated1'regressions''1

12.59045

9TotalPECML-2

B. dose (mg/kg/SO25

12.55025

12.55(1

2512.55025

12.56.9'

5.3'22.0'

66.2'ScheduleQ7DX

2Q4D

x3Q3D

X4Q2DX

5T/C%4.9'

7.6'17.9'17.4'

23.9'56.3'7.6'

10.5'14.1'20.3'1/10

0/100/15

0/10Number

deaths/totaltreated4/10'

0/100/100/10

0/100/100/5

0/50/55/5'

5/5'

0/54/9'

3/10'

0/151/10Complete

regressions5/6'

8/10'3/10'2/10

2/100/100/52/5'

2/5'0/5

°Therapy was initiated 7 days after tumor implant. i.V..daily for 9 days. Five

animals/group/experiment.* T/C% calculated as the mean tumor volume of treated animals on day 21/

mean tumor volume of control animals on day 21. x 100.c Treatment-caused deaths.''Complete regressions at the s.c. site of tumor inoculation were assessed on

day 35. No spontaneous regressions were observed in controls.' Significantly different compared to control groups by Quade rank analysis of

covariance; P set at 0.05.^PEG-IL-2 was administered i.v. starting 7 days after tumor inoculation, on

the schedules shown. The doses were appropriately divided in order to deliver thesame total dose on the various schedules.

based on preliminary single dose studies. A single i.v. dose ofPEG-IL-2 at 50, 25, or 12.5 mg/kg was lethal in 5 of 5, 2 of 5,or 0 of 5 Meth A-bearing mice, respectively. When these sametotal doses were divided and delivered by the various schedules,the toxicity of the treatments was schedule dependent. At 50mg/kg total PEG-IL-2, toxicity was observed only on the Q7Dx 2 and Q2D x 5 schedules. The other combinations of doseand schedule were not lethal, with the exception of the 25 mg/kg dose on the Q2D x 5 schedule (Table IB). As discussedbelow, this dependence on the frequency of administration toproduce the lethality may be related to the minimum plasmaconcentration of IL-2 attained between doses.

The antitumor efficacy of the PEG-IL-2 treatment was alsoschedule dependent; in addition, dose-response relationshipswere observed within each schedule (Figs. 1-3). As shown inTable 1B, the protocol with the best therapeutic index wasfound to be 25 mg/kg given Q7D x 2, in which 8 of 10 completeregressions were produced and no treatment-related deaths wereobserved. A dose dependence was noted on this schedule (Fig.1); however, at the highest dose (50 mg/kg) there were 4 of 10deaths associated with the 5 of 6 complete regressions in thesurviving animals, while at 12.5 mg/kg fewer complete regressions were obtained (3 of 10) without lethality (0 of 10). TheT/C% recorded also reflected this dose dependence. Fig. 1contains the individual hosts' tumor volume measurements

through day 21 on the Q7D x 2 schedule at 50 mg/kg (Fig.1/4), 25 mg/kg (Fig. \B), 12.5 mg/kg (Fig. 1C), and the phosphate-buffered saline controls (Fig. ID). The average tumorvolume of control mice on day 21 was 520 mm3.

The antitumor efficacy obtained on the Q4D x 3 and Q3Dx 4 protocols, as shown by the individual hosts' tumor volume

measurements, is shown in Fig. 2 and Fig. 3, respectively. Oneither of these schedules, the activity was inferior to that obtained on the once-a-week schedule, although it was dose dependent. We hypothesize that this difference may be related to

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DAYS POST IMPLANT

200

180

160

140

120

100

80

B

12 13 14 15 16

DAYS POST IMPLANT

10 11 12 13 14 15 16 17

DAYS POST IMPLANT

19 20 21 22

Fig. 1. PEG-IL-2 antitumor efficacy against Meth A on the Q7D x 2 schedule, individual animal data, through day 21 post-tumor implant. A, Total dose of PEG-IL-2 of 50 mg/kg; B, 25 mg/kg; C, I2.5 mg/kg; D, phosphate-buffered saline controls. The doited line at 20 mm3 is the limit of quantitative tumor volume

determinations.

9 10 1 12 13 14 15 16 17 1

DAYS POST IMPLANT

Fig. 2. As in Fig. 1. on the Q4D x 3 schedule.

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.'0(1

in160

140

120

100

tl

40 t-

.•u-

10 11 12 13 14 15 16 17 18 19 20 21

DAYS POST «PLANT

200

ISO

160

140

120

100

so

«0

40

20

0

10 11 12 13 M 15 16

DAYS POST IMPLANT

B

12 13 14 15 16 17

DAYS POST IMPLANT

18 19 20 21 22

Fig. 3. As in Fig. 1. on the Q3D x 4 schedule.

the kinetics of the IL-2-induced rise in the immune effector cellpopulations. The influence of these biological events, and theirsuperimposition on the pharmacokinetic parameters of scheduling, is an area under current investigation. The high toxicityassociated with the Q2D x 5 schedule precluded the gatheringof meaningful efficacy data on this schedule.

Schedule Dependence in B16. The B16 model was not very-sensitive to rhIL-2 treatment, even at lethal doses, as shown inTable 2A. As in the Meth A studies, the maximum i.v. doseachievable was 112.5 mg/kg total rhIL-2 on the QD x 9schedule; however, in this case, 6 of 16 animals died at thisdose level. The BDF, mice used for B16 were more sensitive torhIL-2 than the BALB/c Meth A hosts at all but the lowestdose; 1 of 16, 5 of 16, and 6 of 16 died at 45, 90, and 112.5mg/kg, respectively. Even at these toxic levels, there were nocomplete regressions obtained in the B16 tumors, and only asmall mean increase of 3.5 days in the tumor growth delayparameter (T—Q was obtained at the highest dose (Table 2).

The antitumor efficacy of PEG-IL-2 was studied at three doselevels, 18, 12, or 6 mg/kg total dose. These levels were lowerthan those studied in Meth A because of the increased sensitivity of the BOP, hosts to its lethal effects, as noted above. Asshown in Table 2B, the toxicity of the treatments was againschedule dependent, in a manner similar to that observed inMeth A. The Q7D x 2 schedule did not produce lethality atthese doses; however, there was a clear dose-dependent lethalityassociated with either the Q4D x 3 or the Q3D x 4 schedule.A single experiment at 30 mg/kg also produced no lethality onthe Q7D x 2 schedule, but no greater efficacy than with 18 mg/kg was obtained. Further studies are ongoing in this model tomore fully establish the doses and length of treatment which

Table 2 rhlL-2 (A) and PEG-IL-2 (B) antilumor efficacy in Hlf> melanoma

TotalrhIL-2A.dose(mg/kg)u112.590459TotalPEG-IL-2doseB.

(mg/kg)'181261812618126ScheduleQ7DX

2Q4D

x3Q3DX4T-C(days)"3.52.92.h1.5T

-C(days)13.6*'10.0''8.1"ND*10.1*7.2"7.2"Number

deaths/totaltreated'6/16"5/16"1/160/16Number

deaths/totaltreated0/160/160/169/10"1/100/1010/10"10/10"6/10"

°rhIL-2 »asadministered QD x 9. i.v.. beginning 1 day after s.c. tumor

implant. Five animals/group in two experiments, six animals/group in the thirdexperiment.

* T —C is the tumor growth delay, determined as the mean number of daysfor the treated tumor volume to reach 500 mm' minus the mean number of days

for the control tumor volumes to reach 500 mm\ Mean value gi\en for threeexperiments.

' Treatment-caused deaths."Significantly different than controls, as in Table 1.e PEG-IL-2 was given i.\. starting 1 day after s.c. tumor implant.•'Threeof six mice in the third experiment had a complete block of tumor

growth through the last day of observation, day 64. These mice were excludedfrom the calculation of T - C.

* Not determined due to excessive toxicity of treatment.

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ENHANCED THERAPEUTIC INDEX OF PEG-IL-2

produce the maximum therapeutic benefit.The efficacy of the PEG-IL-2 on the Q7D x 2 schedule at

nontoxic doses was superior to that obtained with unmodifiedrhIL-2 even when rhlL-2 was administered at toxic levels, asshown in Table 2B by the tumor growth delay parameter T—C.

For example, at 18 mg/kg, a tumor growth delay of 13.6 dayswas obtained (Table 2B), which corresponded to a mean tumorgrowth inhibition of >95% compared to controls at day 28post-implant. In addition, the third experiment in this seriesresulted in a complete block of tumor growth in 3 of 6 micetreated at 18 mg/kg that was durable through day 64, the finalday of observation.

The increase in antitumor activity of PEG-IL-2 is even morestriking if comparisons are made between the efficacy results inTable 2, A and B, at equitoxic dose levels. For example, ateither 9 or 45 mg/kg rhIL-2, a T—C of 1.5 or 2.6 days wasobtained, respectively. PEG-IL-2 treatment at all three dosesof the Q7D x 2 schedule resulted in T-C values ranging from8.1 to 13.6 days, a 3- to 9-fold increase in activity, dependingon the comparison made (P< 0.05). Further, the total doses ofPEG-IL-2 required to obtain these levels of activity were reduced in comparison to those of rhIL-2.

Although the antitumor activity of PEG-IL-2 was also increased on the Q4D x 3 schedule compared to the rhIL-2 atequitoxic dose levels, the lethality observed was greater thanthat observed with Q7D x 2 studies (Table 2B). The excessivetoxicity of the Q3D x 4 schedule resulted in efficacy measurements of limited meaning.

Schedule Dependence in Pan-02. Pan-02 spontaneously me-tastasizes from the s.c. site of inoculation and has shownresistance in vivo to some 50 chemotherapeutic agents (21). Inour hands, it has also proven refractory to several treatmentregimens, including combination therapy studies with chemo-therapeutics and various lymphokines or cytokines. Since thetumor metastasizes, the increase in lifespan (%ILS) of theC57BL/6 hosts can be used as an indication of therapeuticbenefit, as well as the size of the s.c. tumor implant. As a singleagent, rhIL-2 produced limited antitumor efficacy against Pan-02 even at toxic levels when benefit was measured by either ofthese two parameters (Table 3A). The C57BL/6 hosts were alsomore sensitive than BALB/c mice to the lethal effects ofrhlL-2, as was found for BDF, mice.

The three doses of PEG-IL-2 used in the Pan-02 model were12, 6, or 3 mg/kg, as shown in Table 3B. Although the toxicityof PEG-IL-2 was reduced at these dose levels compared tothose used in the B16 model, there was a schedule-dependentincrease in toxicity as the interval between doses was decreased.No deaths were recorded on the Q7D x 2 schedule, 1 of 30mice died on Q4D x 3, 5 of 30 died on Q3D x 4, and 12 of 15died on the three doses of the Q2D x 5 schedule (Table 3B).An additional single study has shown that PEG-IL-2 at 24 mg/kg on the Q7D x 2 schedule also was not lethal.

The antitumor efficacy obtained with PEG-IL-2 was notmarkedly dependent on the schedule of administration, in contrast to the results obtained with the other two models. Theactivity observed at nonlethal doses with PEG-IL-2, however,was again superior to that obtained with rhIL-2 at lethal doses.At the s.c. site of tumor implant, the Q7D x 2 schedule resultedin about a 5-day mean decrease in the rate of growth to 500mm3 (range, 3.2-5.7 days) when compared to controls (T—C)(P < 0.05). The mean T—Con the other schedules ranged fromabout 1.5 to 4 days at nonlethal doses (Table 3B). The Q2D x5 schedule was once again quite toxic and of limited therapeuticvalue.

Table 3 rhIL-2 (A) and PEÜ-lL-2(B) antitumor efficacy in PAN-02

Total rhIL-2 T-CA. dose (mg/kg)" (days)*tíILS*TotalPEG

IL2B.

(mg/kg)'12631263126331.212.52.5112.5

45180.4

0.50.8<100

143'127Number

deaths/totaltreated''2/5'

0/100/5Day

ofdeath*ScheduleQ7DX

2Q4DX

3Q3D

x4Q2D

x 5T

C(days)4.5'5.7'3.24.3'1.92.14.8'2.91.63.5Median30.031.532.536.532.534.538.032.523.515.0Range20->5720-4924-3821-4523-4624-4229-4920-4920-4019-35%ILS153'163'170'197'170'183'207'170'110100totaltreated0/100/100/100/101/100/105/10'0/100/105/5'5/5'0/5

a rhIL-2 treatment started 7 days following s.c. tumor implant, i.v.. QD x 9.

Five mice/group/experiment.* T —C"calculated as in Table 2.c °cILSis the percentage increase in lifespan, calculated as the median day of

death of the treated group/median day of death of the controls, x 100.d Treatment-caused deaths.* Significantly different than controls, as in Table 1.'PEG-IL-2 treatment started 7 days post-implant, i.v., on the schedules

indicated.* Tumor-bearing hosts which did not survive treatment were excluded from

calculations of survival. Days post-tumor inoculation.

OEin

JDQ_

00'c

1000 -c

5.0 10.0 15.0 20.0 25.0

time (hours)

30.0 35.0

Fig. 4. Clearance of rhlI.-2 and PEG-IL-2 following a single i.v. bolus of 7.5mg/kg in mice; IL-2 bioactivity is expressed in Ill/ml plasma. O. rhlL-2; D. PEG-IL-2.

When the treated animals' survival was followed, there were

many instances of an increased lifespan observed, some as muchas 200%. However, there was neither a strong dose nor scheduledependence to this effect, which may reflect the limited numberof animals/group. Nevertheless, the results obtained were encouraging. Additional experiments are ongoing with more prolonged treatment regimens at higher doses, in order to reachthe maximally tolerated dose in this very resistant tumor.

Pharmacokinetics of PEG-IL-2 and rhIL-2 in Mice. Plasmacurves were constructed from the mean concentrations of IL-2bioactivity at each of the time points shown in Fig. 4 for rhlL-2- and PEG-IL-2-treated mice. The prolonged lifetime PEG-IL-2 compared to rhIL-2 suggested that the optimum dosageschedules would produce different plasma concentration pro-

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ENHANCED THERAPEUTIC INDEX OF PEG-IL-2

files for the two proteins. Therefore, the curves shown in Fig.4 were used to predict the plasma curves for some of theeffective dosage schedules used in the efficacy studies.

Both curves were fit to a three-exponential function, withparameter values shown in Table 4 along with the ratio of PEG-IL-2 to rhIL-2 values. Although the plasma curves both extrapolated to the same starting concentration (equal C0 values), themajor difference in the two curves was found in the contributionof the terminal phase of clearance to the overall clearance ofthe proteins. The value of C and the area under the curvecontributed by the T phase (C/r) were increased by 8.23- and30.14-fold, respectively, for PEG-IL-2. The half lives for allthree phases were longer for PEG-IL-2, and the total area underthe plasma curve (AUC) for PEG-IL-2 increased 11.76-foldover that of rhIL-2. The parameters from Table 4 were used tosimulate plasma curves for the four PEG-IL-2 schedules whichwere studied in the Meth A efficacy model at 25 mg/kg (Fig.5).

The simulations shown in Fig. 5 demonstrate that peakconcentrations among the schedules for PEG-IL-2 ranged from0.6 to 1.6x IO6lU/ml, less than 3-fold. However, the minimum

concentration achieved between doses or the duration below 10lU/ml between doses varied greatly among the schedules andappeared to correlate with toxicity. For example, when the 25mg/kg total dose was given Q7D x 2 the time below 10 lU/mlwas almost 4 days, whereas the Q4D x 3 schedule reduced thistime to 1 day and the Q3D x 4 schedule further reduced thistime to only a few hours. The Q2D x 5 schedule produced aminimum concentration between doses of 94 ID/ml and was100% lethal in the Meth A model (Table IB). For rhIL-2 givenat 12.5 mg/kg/day (approximately the LD,0), the daily scheduleallowed about 6 h/day below 10 lU/ml.

DISCUSSION

In the Meth A, B16, and Pan-02 tumor models, the antitumorefficacy of PEG-IL-2 was found to be superior to that of rhlL-2 at equitoxic doses. Further, in these three murine tumormodels the toxicity and the antitumor efficacy of PEG-IL-2was found to be dependent on the schedule of administration(Tables IB, 2B, and 3B). The Q7D x 2 schedule was the least

Table 4 Pharmacokinelk parameters in mice given 7.5 mg PEG-IL-2 or IL-2/kgMale CD mice were dosed once i.v. with 7.5 mg/kg rhIL-2 or with 0.25 mg/

kg PEG-IL-2. Five mice/time point, times of collection are shown in Fig. 4. Oneplasma sample was collected from each mouse by rctroorbital sinus puncture. IL-2 bioactivity. measured by the methods of Gillis el al. (22) and Watson (23). wasadjusted for dose.

Parameter"A,

(KU/ml)B, (KU/ml)C(KU/ml)C0.

(KU/ml)r(V,,o

(min)r(Ä„(min)7",»,,(min)A/a.

|(KU/ml)h]lì/,1.l(KU/ml)h]C/r,|(KU/ml)h)AUC,

|(KU/ml)h|PEG-IL-2656

21962.093710.0

82.8327.9158

436489108.1rhll.-2769

1497.539251.612.9

89.429.7

46.116.292.0PEG-IL-2/rhIL-20.85

1.478.231.016.25

6.423.675.32

9.4530.1411.76

" Parameters »eredefined as follows: A, B. and fare the zero-time interceptsof each clearance phase obtained from curve-stripping: C0 is the zero-timeintercept of the plasma curve; the TV,values (half lives) were calculated from-ln(0.5)/ratc constant; A/a. B/ii. and C/r are the areas under each phase of theplasma curve; and AUC is the area under the plasma curve from time zero toinfinity.

toxic at the doses studied and resulted in the best antitumoractivity. More closely spaced dosing regimens were increasinglytoxic as the interval between doses was decreased. Further, noenhancement of the efficacy was obtained by decreasing theinterval between doses and in many instances the antitumoractivity was reduced in comparison to the Q7D x 2 schedule.

Meth A was somewhat sensitive to rhIL-2, as 3 of 10 and 4of 10 complete regressions were obtained at the two highestdose levels. The PEG-IL-2 was more active, however, as 8 of10 complete regressions were observed at equitoxic, or evenless than equitoxic, doses (Table 1). In B16, which was marginally sensitive to rhIL-2 even at lethal dose levels, PEG-IL-2treatment at nonlethal doses resulted in a 2-fold increase intumor growth delay (T—C)compared to rhIL-2 at lethal doses

(Table 2). If the highest nonlethal doses were compared, anincrease of over 9-fold in T—Cwas obtained with PEG-IL-2treatment: T-C of 13.6 days for PEG-IL-2 Q7D x 2 at 18 mg/kg, compared to 1.5 days for rhIL-2 at 9 mg/kg, QD x 9. Acomplete block of tumor growth in 3 of 16 treated animals wasalso observed with PEG-IL-2 at this dose. In Pan-02, a tumorresistant to rhIL-2 treatment, PEG-IL-2 increased the T—C toas much as 5.7 days (6 mg/kg, Q7D x 2). In addition, anincrease in the lifespan of the tumor hosts of from 15090 tonearly 200% was obtained with PEG-IL-2 treatment at nonlethal doses (Table 3).

The response of other methylcholanthrene-induced fibrosar-comas (5) and of murine mammary adenocarcinomas (8, 12) torhIL-2 or rhIL-2 in combination with lymphokine-activatedkiller cells (3, 4) has been found to correlate with the degree ofimmunogenicity of the tumor. Meth A has also been shown tobe immunogenic (25), which probably contributed to its sensitivity to rhIL-2 and PEG-IL-2. The B16 and Pan-02 tumorsapparently are nonimmunogenic, however, as defined by reim-plantation studies and as confirmed by their resistance to rhlL-2. The PEG-IL-2 treatments did result in a significant amountof antitumor activity, however, which suggested that perhapseven resistant nonimmunogenic tumors could respond to IL-2if a more optimum manner of therapy was used. It should alsobe clear that for these more resistant tumors, two weeklytreatments may not be the most therapeutic schedule possiblebut it was chosen to match those used in Meth A in order todevelop the comparisons made in this series of experiments.Additional weekly treatments might increase the degree ofantitumor activity obtained in these more resistant models.

In earlier studies with a PEG-IL-2 species modified to alesser extent that the material used in the present studies, fivedaily i.p. treatments of 80 Mg/kg (0.4 mg/kg total dose) resultedin a marked decrease in the growth of Meth A at day 10 (2).Additional investigations revealed that this schedule and routewas a comparatively toxic method of PEG-IL-2 administration.Therefore, we began to investigate the protocols described inthe present study to establish less toxic and more efficaciousregimens. In addition, pharmacokinetic studies have demonstrated that modification of rhIL-2 with PEG prolonged thehalf-life in proportion to the degree of modification of the rhlL-2 and that the clearance can be predicted from the hydrody-namic radii of the modified species (1). We hypothesi/.ed fromthis work that perhaps different degrees of modification wouldsufficiently alter the pharmacokinetics such that determinationof the protocol with the best therapeutic index would dependon these clearance characteristics. Preliminary /'/; vivo tumor

studies comparing a PEG-IL-2 species with apparent molecularweight of 93,000-95,000 with the results presented here haveconfirmed these expectations.

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ENHANCED THERAPEUTIC INDEX OF PEG-IL-2

Fig. 5. Pharmacokinetic simulations ofPEG-IL-2 in mice dosed i.v. with 25 mg/kgtotal dose on the four schedules studied in theMeth A model; IL-2 bioactivity is expressed inlU/ml plasma. A, Q7D x 2 schedule (12.5 mg/kg/dose); B, Q4D x 3 schedule (8.33 mg/kg/dose); C. Q3D x 4 schedule (6.25 mg/kg/dose); D. Q2D x 5 schedule (5 mg/kg/dose).

a,0.

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The toxicity of PEG-IL-2 is hypothesized to be related to theminimum plasma concentration attained between doses, asillustrated by the striking schedule dependency of PEG-IL-2

toxicity in these studies (Tables IB, 2B, and 3B). In addition,the duration of low plasma concentrations also appeared to beimportant. For example, at 25 mg/kg on the Q7D x 2 schedule(a nontoxic regimen in Meth A), the calculated minimumplasma levels reached approximately 6 x 10~5 lU/ml, a theo

retical number of questionable biological significance. In addition, however, the interval between which measurable levels (1-10 lU/ml) could be reliably detected was about 4 days, thelongest of the schedules tested (Fig. 5^).

On the Q3D x 4 schedule at 18 mg/kg or 12 mg/kg, toxicdeaths were recorded in the B16 (10 of 10) or Pan-02 (5 of 10)models, respectively. The minimum plasma levels in this casewere approximately 6 ID/ml, which meant there was no timeinterval during which the PEG-IL-2 levels were below measurable concentrations (Fig. 5C). The Q2D x 5 schedule provedto be essentially 100% lethal; in this case, the lowest levelsachieved between doses were about 94 ILJ/ml (Fig. 5D).

Further, high single doses of PEG-IL-2 were toxic, whichalso suggested that the time IL-2 stays above a particular plasmaconcentration can contribute to toxicity. Data obtained usingan unmodified rhIL-2 with improved solubility have indicatedthat 75 mg/kg as a single i.v. bolus is not lethal in Meth Atumor-bearing BALB/c mice. The clearance of this material isvery similar to the rhIL-2 shown here. Therefore, these dataalso support the hypothesis that the peak plasma concentration

alone does not determine the toxicity of IL-2 but that theclearance characteristics are also major contributory factors toIL-2 toxicity.

The efficacy obtained is hypothesized to be related to thepeak plasma IL-2 concentration, as well as the manner in whichthe levels fall off, since in all three tumor models there was adose dependence to the efficacy obtained. The Q7D x 2 schedule resulted in the best therapeutic index, since there was littleor no lethality associated with the treatments and substantialantitumor activity was observed (Tables IB, 2B, and 3B). Thedegree of antitumor activity obtained was also schedule dependent, however, which suggested that while higher peak IL-2concentrations improved the antitumor response the doses hadto be delivered at appropriate intervals in order to reduce thetoxicity of the treatment. One pharmacokinetic factor that didnot appear to be significant was the area under the curve. Sincethe same total dose was delivered on each of the differentschedules, the area under the curve would be equivalent at eachdose level.

While the treatment schedule and pharmacokinetic parameters appeared to be important factors in the therapeutic responses obtained, one must also consider the hosts' biological

responses to IL-2 and the kinetics of these responses. We areinvestigating the hypothesis that the once-a-week treatmentschedule was superior to the other schedules in part due to acoordination of these biological responses with the PEG-IL-2administration. For example, it has been shown in clinical trialsthat the peripheral WBC population increases dramatically (up

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ENHANCED THERAPEUTIC INDEX OF PEG-IL-2

to 16-fold) on days 6-8 following 5 days of rhIL-2 therapy(Refs. 13-19 and references therein). It may be that the Q7Dx 2 schedule takes advantage of such cycles, which are poorlydefined at this time, either to enhance the efficacy or reduce thetoxicity of PEG-IL-2. In addition, there may be subtle differences in the pharmacokinetics or biological consequences ofPEG-IL-2 in tumor-bearing animals, compared to non-tumor-bearing, or in different strains of mice. Studies to address therelationships between these biological responses and the treatment schedules are ongoing in order to more fully understandtheir impact on the toxicity and antitumor efficacy of PEG-IL-2.

ACKNOWLEDGMENTS

The authors thank Dick Bell, Tony Chan, Stacey Gauny, PhoebeLandre, Ping Luo, and David Wong for excellent technical assistanceand Sharon Nilson for illustrations.

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1989;49:6521-6528. Cancer Res   Robert J. Zimmerman, Sharon Lea Aukerman, Nandini V. Katre, et al.   ModelsPolyethylene Glycol-modified Interleukin 2 in Murine Tumor Schedule Dependency of the Antitumor Activity and Toxicity of

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