Download Importance of Treatment Regimen of Interferon

[CANCER RESEARCH 43, 4172-4175,
September 1983]
Importance of Treatment Regimen of Interferon as an Antitumor Agent1
S. H. Lee, H. Chiù,E. Rinderknecht, W. Sabo, and N. Stebbing
Genentech, Inc., South San Francisco, California 94080
ABSTRACT
A highly purified hybrid human leukocyte Interferon, IFN-aAD,
produced in Escherichia coli, has been used to define optimum
treatment conditions for L1210 leukemia in mice. Treatments
prior to tumor inoculation were ineffective. Treatments from the
third day post-tumor inoculation were most effective, and treat
present study, we have investigated various treatment regimens
with highly purified IFN-aAD in L1210 tumor-bearing mice with
the objective of defining optimum regimens so that significant
mechanisms of action may be identified.
MATERIALS AND METHODS
ments every third day were more effective than were regimens
involving more frequent treatments. IFN-aAD was effective in
Interferon. The preparation of IFN-aAD has been reported elsewhere
(28). IFN-aAD was purified from Escherichia coli lysates by a combination
vivo against tumors formed from a line of L1210 cells resistant
to IFN-aAD in cell cultures. These and other results indicate the
of methods described previously (26, 30). The purified material was
homogeneous by polyacrylamide gel electrophoresis and had a specific
activity of 1.9 x 108 units/mg protein when titrated on HeLa cells against
importance and nature of indirect mechanisms
efficacy of interferons against tumors.
of action for
INTRODUCTION
Antitumor activities of interferons have been attributed to a
number of effects observed in cell cultures. These include direct
antiproliferative activity as well as indirect activities such as
stimulation of NK2 cell activity, antibody synthesis, and antibodymediated cell cytotoxicity (2, 5-7, 10-13, 15, 27). However,
antitumor effects have been observed in mice bearing tumor
cells resistant to antiproliferative effects of interferoni (14). Al
though stimulation of NK cell activity has been observed in clinical
studies (3), a recent report indicates decreased rather than
increased NK cell activity after Interferon treatment (17). The
contribution of mechanisms assayed in cell cultures to in vivo
antitumor effects is further confounded by use of impure interferon preparations and observations that opposing effects may
occur depending on the interferon concentration used and the
timing of treatments (4, 18). Generally, mechanisms of action
have been assumed from in vitro assays without regard to how
the parameters measured are affected in optimum treatment
regimens.
Cloning of human interferon genes into bacteria has led to
production of highly purified materials, such as the human leu
kocyte interferon subtypes, interferon-aA and interferon-aD (pre
viously designated LeIF-A and -D) (8, 9).
The existence of common restriction enzyme sites in the
various leukocyte interferon genes has allowed production of
novel molecular hybrid interferons, such as IFN-aAD (28), which
consists of the 61 NH2-terminal amino acids of interferon-aA and
the 104-amino acid COOH-terminal portion of interferon-aD. IFNaAD is remarkable for its high activity on mouse cells as well as
human cells (16, 29), and this interferon also shows antiviral
activity in encephalomyocarditis virus-infected mice (29). IFNaAD appears to have essentially the same specific activity on
mouse cells as purified mouse interferon preparations.3 In the
1This work was supported by Genentech, Inc., South San Francisco, Calif.
2The abbreviations used are: NK cell, natural killer cell; IFN-aAD, hybrid human
leukocyte interferon composed of the 61 NhMerminal residues of subtype IFN-aA
and the 104 COOH-terminal residues of subtype IFN-aD.
3 C. Czamiecki, unpublished results.
Received March 11,1983; accepted June 7,1983.
4172
the NIH human leukocyte interferon standard (G-023-901-527).
All inter
feron titers are given here in terms of the ID.
Mice. Adult (8 to 10 weeks old) female C57BL x DBA/2 F, (hereafter
called BD2F,) mice used for all experiments were purchased from Charles
River Laboratories, Wilmington, Mass. Twenty mice/group were inocu
lated with u 210 cells i.p. for all experiments.
Cells. L1210 cells were serially passed and maintained by weekly
inoculation of 1 x 106 cells into the peritoneal cavity of DBA/2 mice
(Charles River Laboratories).
RESULTS
A daily dose of 10s units IFN-aAD given i.p. for the 5 days
preceding administration of L1210 tumor cells to mice had no
effect on survival of the mice. Similarly, treatments during the
first 2 days after inoculation of tumor cells caused no significant
effect on survival. However, if treatments were extended to later
times after tumor inoculation, significant cure was observed. The
data in Chart 1 show that daily treatments from the third day
post-tumor inoculation appear to be more effective than treat
ments commencing 1, 5, 7, or 9 days post-tumor inoculation.
These treatments were given daily until the 12th day post-tumor
inoculation, and all the treatment regimens conferred significant
protection (p < 0.05) except the one commencing at 9 days
post-tumor inoculation. Logrank x2 analysis (21) showed that
the regimen commencing 3 days post-tumor inoculation was
more effective (p < 0.05) than any of the other treatment
regimens. The relatively poor effect of the regimen commencing
9 days post-tumor inoculation must be due in part to the fact
that only 3 treatments were given in this regimen and some
deaths occurred before treatment commenced. However, we
conclude that treatments commencing on the third day posttumor inoculation are most beneficial. These studies were ex
tended to examination of the effects of frequency and duration
of treatments. Treatments commencing 3 days post-tumor in
oculation and then given daily, or every second, third, or fourth
day, all conferred protection (Chart 2a) which was significant in
each case when compared to the control group. Treatments
every third day conferred the greatest protection, and this was
significantly greater than that conferred by treatments given daily
or every fourth day. The relatively poor effect of treatments every
fourth day may be due in part to the fact that only 4 treatments
were administered before deaths occurred. However, 7 treatCANCER
RESEARCH
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VOL. 43
Antitumor Activity of Leukocyte Interferon
Days post tumor inoculation
Chart 1. Effect of IFN-aAD (105 units/treatment) on L1210 leukemia in BD2F,
mice. The L1210 cell strain used was one initially developed by Pincus et al, (22),
and the cells were passaged in DBA/2 mice. All treatments were given once daily
i.p. up to the 12th day post-tumor inoculation with treatments commencing on Day
1 (O), Day 3 (: !). Day 5 (A), Day 7 (A), or Day 9 (•)post-tumor inoculation. •.
untreated control group, 10* L1210 cells/mouse i.p. Numbers in parentheses,
median survival time in days.
concentrations were no longer detectable. The circulating interferon must represent that injected into the animals and not
induced mouse interferon because the assay was positive on
human WISH cells as well as on mouse L-929 cells (28). In L1210
cultures, IFN-aAD at concentrations of 2,500 and 10,000 units/
ml caused 40 and 50% inhibition of the rate of growth, respec
tively, measured by the number of viable L1210 cells after 3
days of incubation in the presence of IFN-aAD. These observa
tions indicate that direct effects of IFN-aAD may play a role in
antitumor effects.
In order to assess the importance, if any, of direct inhibition of
L1210 cells by IFN-aAD in vivo, a line of the L1210 cells resistant
to the growth-inhibitory effects of IFN-aAD was developed. This
was achieved by continuous passage in culture in the presence
of 10" units/ml IFN-aAD over a period of 10 weeks. Surviving
cells were cloned in agar, and one clone was selected in medium
containing IFN-aAD (3 x 104 units/ml) and resistance has now
persisted for more than 10 passages after removal of the inter
feron. This interferon-resistant cell line causes death of BD2F!
mice at 106 cells/mouse, but the mean time of death is 30 to 35
days. However, mice treated 7 times with IFN-aAD at 48-hr
intervals from 1 day post-tumor inoculation caused significant
protection against the resistant line of L1210 cells (Chart 3).
Maximum inhibition of in vitro plaque-forming cells is known
to occur when animals are given interferon before antigenic
stimulation (sheep RBC), and enhancement occurs when inter
feron is given after 2 or 4 days (4). Thus, interferon treatments
from the day of tumor inoculation or earlier could suppress
development of an immune response, whereas treatments from
the third day post-tumor inoculation could enhance antitumor
antibody production and cure mice bearing the L1210 leukemia.
If this is the case and antibody response is necessary for
antitumor effects of IFN-aAD, then treatment of mice with IFNaAD before tumor inoculation should abrogate the efficacy of
treatments given after tumor inoculation. This was found to be
the case. Treatment of BD2F, mice with IFN-aAD prior to tumor
inoculation decreased the antitumor activity of IFN-aAD given
post-tumor inoculation. Mice which received one injection of IFNaAD (10s units) 48 hr prior to tumor inoculation had a mean
io
I5
Days post tumor inoculation
Chart 2. Effect of IFN-aAD (10s units/treatment) on L1210 leukemia in BD2F,
mice with treatments (a) at various frequencies or (b) for various numbers of days.
All treatments were given once daily i.p. commencing on the third day after tumor
inoculation, a, treatments given 7 times, daily (O), every second day (A), every third
day (LI), or every fourth day (A), b. all treatments given daily for 1 day (A), 3 days
(D), 5 days (A), 7 days (•),9 days (O), or 12 days (T). •,untreated control group,
10* L1210 cells/mouse i.p. Numbers in parentheses, median survival time in days.
survival time significantly (p < 0.05) shorter than did mice which
were not pretreated (Chart 4).
DISCUSSION
It should be noted that the initial strain of L1210 cells used in
this study (22) results in a mean survival time at 106 cells/mouse
of about 10 days. At lower tumor doses (5 x 103 cells/mouse),
the mean survival time of control groups is 15 to 20 days, and
ments on consecutive or alternate days were certainly no more
effective.
The effect of duration of treatments was examined by giving
daily treatments from the third day post-tumor inoculation. The
regimens examined were a single treatment and treatments for
3, 5, 7, 9, or 12 days. The results, in Chart 2o, show that
prolongation of survival is proportional to the duration of treat
ment. Only the 2 shortest treatments failed to confer significant
protection.
Measurement of blood concentrations of IFN-aAD after i.p.
administration of 105 units/mouse showed that the maximum
titer was 2500 units/ml, and this occurred after 30 min. Clearance
from the circulation occurred over 4 hr, at which time blood
SEPTEMBER
IO
15
20
25
30
ÃŽ5
50
Days post tumor inoculation
Chart3. In vivo effect of IFN-aAD (106 units/treatment) on L1210 leukemia
resistant to IFN-aAD. D, treatments given 7 times every second day commencing
on Day 1. •,untreated control group, 10°resistant L1210 cells/mouse i.p. Number
in parentheses, median survival time in days.
1983
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4173
S. H. Lee et al.
io
15
20
25
30
Days post tumor inoculation
35
40
45
Chart 4. Effect of a pretreatment with 10* units IFN-aAD at -48 hr on L1210
leukemia in BD2F, mice. Treatments of 10s units given 7 times every second day
commencing on Day 3 post-tumor inoculation, without pretreatment (D) or with the
pretreatment (•).Control group, 10* L1210 cells/mouse i.p. without any interferon
treatments (•)and with the pretreatment
survival time in days.
(A). Numbers in parentheses,
median
the majority of mice are cured by the IFN-aAD treatment regi
mens described here. The larger tumor dose was used in the
present studies in order to assess the comparative efficacy of
various treatment regimens. It is possible that the nature of the
tumor cell line and the greater mean survival time that occurs
with this L1210 strain in BD2Ft mice may influence the effects
observed.
Although more effective treatment regimens might be devised,
the data presented here indicate that efficacy of IFN-aAD against
the L1210 leukemia is greatest with treatments given every
second or third day commencing after tumor inoculation. The
doubling time of the L1210 cells used was approximately 12 hr
for the initial cell line and 16 hr for the line resistant to IFN-aAD.
A lag time of about 24 hr was observed in vivo for both cell lines.
A tumor cell kill of at least 90% is expected to cause a delay in
death of only 2 days (25). Much greater delays in mean survival
time were observed in the present studies with an agent, IFNaAD, which causes less than 50% inhibition of growth of the
tumor cell in culture at the blood concentrations achieved after
i.p. treatment of mice with 105 units. Moreover, the line of L1210
cells resistant to growth-inhibitory effects of IFN-aAD caused
tumors in mice which still responded to IFN-aAD treatment. To
assure the resistant phenotype of the L1210 cells after in vivo
passages, the growth-inhibitory effect of IFN-aAD was tested
against these cells after 3 in vivo passages. The result of this
experiment showed that these cells retained complete resistancy
at 3 x 10* units of IFN-aAD per ml. Thus, in vivo instability of
between IFN-aAD-treated and untreated control mice was ob
served. The antitumor effects are most pronounced with treat
ments from the third day after tumor inoculation, which indicates
the need for development of some response to the tumor. This
could be an immune response to the tumor. However, any
circulating antibodies mediating such effects are likely to be short
lived or require other factors for efficacy because cured mice
were found to be susceptible to reinoculation of L1210 tumor
cells (data not shown). Several papers have been reported in the
literature that interferons also enhance antibody-dependent cy
totoxic cell activity (1,19) and activate macrophage tumoricidal
activity (23, 24). Since both of these effector functions require
circulating antibody for maximal tumoricidal activity (20), it is
conceivable that antitumor activity is the result of antibodydependent cell-mediated cytotoxicity enhanced by IFN-aAD. Fur
ther investigation is necessary to determine the importance and
extent of involvement of these effector functions. If specific
immune responses play a role in controlling tumor development,
then the effects of interferons on antibody responses could be
important. For effective clinical use, optimized treatment regi
mens now need to be investigated further to determine mecha
nisms of primary importance for antitumor effects.
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VOL. 43
Antitumor Activity of Leukocyte Interferon
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1983
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4175
Importance of Treatment Regimen of Interferon as an Antitumor
Agent
S. H. Lee, H. Chiu, E. Rinderknecht, et al.
Cancer Res 1983;43:4172-4175.
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