Download Enhanced Antitumor Activity of Combination

[CANCER RESEARCH57. 2181—2186,
June I, 1997J
Enhanced Antitumor Activity of Combination Radioimmunotherapy
Monoclonal Antibody A33) with Chemotherapy
Jörg Tschmelitsch,
Els Barendswaard,
Clarence
Williams,
(1311-labeled
(Fluorouracil)'
Jr., Tzy-Jyun
Yao, Alfred
M. Cohen,
Lloyd J. Old, and
Sydney Welt2
New York Branch, Ludwig institute for Cancer Research IE. B., C. W., L J. 0., S. W.j, Department of Surgery If. T., A. M. CI, and Department of Epidemiology
IT-f. Y.J, Memorial Sloan-Kettering Cancer Center, New York, New York 10021
analysis showed significant survival benefit for adjuvant 5-FU + LV
ABSTRACT
Monoclonal antibody (mAb) A33 reacts with an antigen expressed by
>95% of colon cancer and normal colon epithelial cells. An earlier Phase
I trial of ‘3'I-labeled
mAb A33 (‘3'I-mAb
A33) demonstrated bone mar
row suppression as the dose-limiting toxicity, and although modest anti
tumor effects were seen, no normal colon toxicity was observed. In this
study,
a nude
mouse
model
was
used
to test
whether
combinations
of
low-dose ‘311.mAb
A33 (0.1 mCI) and chemotherapy [5-fluorouracil (5FU) or 5-FU + leucovorin, doxorubicin, or carmustine] enhance the
antitumor effects, compared to ‘311-mAb
A33 alone or either drug regi
men alone. 5-FU was administered either at 30 mg/kg/day for 5 days or at
75 mg/kg/day on days 1 and 5. In assessing the reduction in tumor volumes
over the first 28 days of the experiment, 5-FU treatment (with or without
leucovorin) in combination with ‘3m1-mAb
A33 showed a statistically
significant additive antitumor effect compared to ‘311-mAb
A33 alone or
to chemotherapy alone. When long-term survival was used as an end
point, 38% of the mice treated with 5-FU and ‘311.mAb
A33 were disease
free at 276 days comparedto none from any other group,suggestinga
synergistic effect. These data indicate that Phase II clinical trials combin.
Ing radlolabeled antibody therapy with 5-FIJ-based treatments are war
ranted.
@
INTRODUCTION
Cancer of the colon and rectum is the second most common cancer
in theUnitedStates(1).Surgeryis thecornerstoneof treatment,but
surgeryalonehas a highrecurrencerate evenin the treatmentof
primary, localized disease, Only lately have adjuvant chemotherapy
regimens for colon cancer, or adjuvant chemoradiotherapy regimens
for rectal cancer, shown an impact on survival (2—6),
5-FU,3 the mainstay ot' treatment, blocks the activity of thymidylate
synthetase and/or is activated to fluoro-UTP and incorporated into the
RNA, However, the efficacy rate of 5-FU is limited (7—9).Recently,
many attempts have been made to increase the antitumor activity of
t'Iuoropyrimidine using biochemical modulators. LV is one such mod
ulator, and its mode of action is considered to be an increase in
thymidylate synthetase inhibition through formation of a ternary com
plex of 5, 10-methylene tetrahydrot'olate from LV, thymidylate syn
thetase, and t1uoro@dUMP (10—12),
Whether the strategy of 5-FU modulation by LV is clinically
significantremainsunclear,SomePhaseIIIclinicaltrialshaveshown
that 5-FU ±LV provides a significantly higher response rate than
5-FU alone ( 13—16),and a recently published multicenter, pooled
Received 7/29/96; accepted 4/7/97.
The costs of publication of this article were defrayed in part by the payment of page
charges. This @rtjcI@
must therefore be hereby marked atl@'eriiseit,ei,t ifl accordance with
18 U.S.C. Section 1734 solely to indicate this fact.
â€T̃his
study
was
supported
in part
by
funds
provided
by
Prof.
F. Bodner,
CA-05748 from the National Cancer Institute.
whom
requrstu
for
reprints
should
Research, Memorial Sloan-Kettering
10021-6007.
3 The
abbreviations
used
are:
5-FU,
be
addressed,
at
Ludwig
Institute
for
treatment
after colon resection
( 17). However,
other studies
have not
shown a significant benefit (18, 19). Even under these improved
conditions,
the response
rate is at best only
35%.
Therefore,
new
therapeutic approaches are needed to treat this type of cancer.
Another approach to enhance the efficacy of 5-FU is to take
advantage
of the additive (20, 21) or even synergistic
effect of the
combination
of 5-FU and radiation
(22—25), which is believed
to
account in part for better control of local recurrences of rectal cancer.
In primary or advanced colon cancer and advanced rectal cancer,
external beam radiation therapy is not applicable for technical reasons
and because of adjacent radiosensitive organs. Radiolabeled mAbs
targeting
colon
cancer
might
be able to deliver
the needed
radiation
dose precisely and without major toxicity. A Phase I/I! ‘3'I-mAbA33
therapy trial demonstrated bone marrow as the dose-limiting organ
toxicity (26). Due to the localization of radiolabeled antibody to
normal colon, gastrointestinal toxicity was examined closely and was
found to be minimal. Antitumor effects were observed in 5 of 23
assessable patients. despite the fact that only a single dose could be
administered due to development of an antimouse immunoglobulin
response (26). To determine toxicity, treatment schedule, and thera
peutic efficacy of a combined approach, different combinations of
low-dose (0, 1 mCi) ‘‘I-mAbA33 and chemotherapeutic agents were
evaluated in a nude-mouse, human colon cancer-xenograt't model.
Because the radiation dose delivered to solid tumors by radiolabeled
antibodies is low in clinical studies (27), we selected a subtherapeutic
dose of ‘311-mAb
A33 for this study to search for enhanced antitumor
effects at low radiation dosages,
MATERIALS
AND METHODS
Cell Lines. Humancolon carcinomacell line SWI222 was obtainedfrom
the cell bank of the Ludwig Institute for Cancer Research at Memorial
Sloan-Kettering Cancer Center and maintained in Eagle's MEM containing 1%
nonessential amino acids and supplemented with 10% FCS, 2 mM glutamine,
100 units/mI penicillin. and 100 @g/mlstreptomycin. Cells were cultured in a
37°Cincubator containing 5% [email protected] cells were harvested using 0. 1%
trypsin and 0.02 EDTA (Life Technologies,
Inc., Grand Island, NY).
Human Tumor Xenografts, Four- to 6-week old female Swiss (nu/nu)
athymic mice (20—25g body weight) were obtained from the Memorial
Sloan-Kettering Cancer Center nude mouse facility and injected i.m. with
10 x io@cells in 250 @i1
PBS into the left thigh muscle. After I week, mice
bearing tumors weighing approximately 100—5(X)
tug were selected. Mice
were anesthetized with 0.5 ml Avertin (2,2,2-tribromoethanol, 97%; PlaIts and
Bauer, Waterbury, CT) by i.p. injection.
Labeling of mAim, lodination of mAbs was carried out using the chlor
amine T method. mAb A33 and mAb FB5, an lgG2a against human neovas
cular endothelium used as an isotype control, were mixed with the requisite
quantity
Chairman,
Second Department of Surgery, University of Innubruck, Innsbruck, Austria, and Grant
2 To
and Biostatistics
of ‘@‘i
(1 mg/20
mCi). Two hundred
@Ll
of a freshly
prepared
solution
of 2 mg/mI chioramine T (in 150 mM sterile phosphate buffer at p1-17.4) were
added to the mixture. After I mm, the reaction was quenched by the addition
Cancer
Cancer Center, 1275 York Avenue, New York, NY
of 200 @ilof a freshlypreparedsolutionof 10 mg/mlsodiummetabisulfite.
Labeled protein was separated from the free iodine on a Sephadex 025 column
5-fluorouracil;
LV,
leucovorin;
mAb,
monoclonal
antibody; ‘3'l-mAbA33, ‘3'I-labeled
mAb A33; ‘3'J-mAb
FBS, ‘3'I-labeled
mAb PBS;
MTD,maximumtolerateddose;AUC,areaunderthe fittedgrowthcurve;% 101g.% of
injecteddose/g;MTS,meantumorsi@c.
(Pharmacia),saturatedwith 0.5% BSA/PBS.Antibodyfractionswith peak
radioactivity
were pooled,
and aliquots
of the ptoled
fractions
were tested
for
the percentage of precipitable protein-bound ‘@‘i
by the trichioroacetic acid
2181
Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1997 American Association for Cancer Research.
COMBINATION RADIOIMMUNOTHERAPYAND CHEMOTHERAPY
precipitation
method.
The amount
of trichloroacetic
acid-precipitable
Table 1 MeancurveTreatment
and SE of the area under the fitted logarithmic tumor gr
protein
bound ‘@‘I
was 97—99%.
Immunoreactivity for mAb A33 was determined as described previously
(28) by adding 0.1 @Ci
of ‘311-mAb
A33 to 15 X 106 antigen-positive cell
pellets. To determine background cpm, cell pellets were pretreated with
>100
X excess unlabeled
Immunoreactivity
from day 0 to day 28owth
day)SEExperiment
20.12.55-FUFB5
—1.61.95-Ri
bolus
mAb A33 prior to addition of ‘311-mAbA33.
was calculated
by subtracting
background
cpm from
AUC (ln (cm3) X
I
13'I-mAb
—48.36.0@3'I-m@b
A33
‘@‘I
—73.411.35-FU
bolus + ‘3'I-mAbA33
mAb A33 cpm bound to the cell pellet, after washing twice in PBS and
dividing the remaining fraction by the total cpm added. Mean immunoreac
—7.82.05-FU
bolus + ‘3'I-mAbFB5
daily
tivity was 34%. A single dose of 0.1 mCi of ‘3'I-mAbA33 or ‘3'I-mAbFB5
5-nj daily+ ‘3'I-mAb
A33
—77.7
5-FU daily + ‘311-mAbFBS
2.1Control
—3.42.5
in 0.2 ml of PBS was injected
iv. into the retro-orbital
plexus.
Formulation and Administration of Drugs. 5-FU (50 mg/mI;SoloPak
—4.8
25.53.9Experiment
2
Laboratories, Franklin Park, IL) was obtained as a pharmaceutical preparation
—38.34.6s-at
‘31I@bA33
and diluted to a concentration
—12.83.95-nj
+ LV
—53.84.05-FU
+ LV + ‘3'I-mAb
A33
of 5 mg/ml or 10 mg/mI in sterile 0.9% saline.
It was injected i.p. either at 30 mg/kg/day for 5 days or at 75 mg/kg/day on
days
1 and 5. The injected
volume
ranged
from 0.12 to 0.19 ml.
9.0
16.73.4Experiment
+ LV + ‘31I-mAbFB5
—
3
—37.93.85-FU
t311-mAbA33
LV (Burroughs Wellcome Co., Research Triangle Park, NC) was dissolved
—74.44.7Doxorubicin
+ LV + ‘311-mAbA33
in 0.9% sterile saline at a concentration of 10 mg/mI. Mice treated daily
received 90 mg/kg LV i.p. 2 h prior to 5-FU. The injected volume was 0.18
ml/20 g body weight.
Doxorubicin (Sigma Chemical Company, St. Louis, MO) was given i.p. as
a single dose of 10 mg/kg. Carmustine (Bristol Myers Squibb, Princeton, NJ)
was injected i.p. as a single dose of 30 mg/kg. Both drugs were dissolved in
—39.96.1Doxorubicin
+ 1311-mAbA33
1.05.3Carmustine
+ ‘
3'I-mAbFB5
—
—27.04.8Carmustine
+ ‘3'I-mAbA33
15.64.2‘3'I-m@b
+ ‘311-mAb
FB5
20.22.5Control
FB5
26.45.3
PBS sufficientto yield a volume thatpermittedadministrationin a dose of 0.1
mi/lO g body weight and injected on day 1.
The doses used were selected from the literature and first tested in tumor
bearing animals in preliminary toxicity studies to determine the MTh.
Tumor Therapy. Mice were divided into groups of six to eight animals.
Toxicity
was determined
by measuring
change
in body
weight.
All animals
were weighed at the start of the experiments and at 8-day intervals. Tumor size
@
was determined by digital caliper measurement. Two perpendicular diameters
were used to calculate tumor volumes (29). Measurements were taken imme
diately before therapy and at 4-day intervals until day 68 or until tumor volume
reached 2 cm3 (2 g).
In the first experiment, one group of mice each received 0. 1 mCi of either
‘311-mAbA33 or control ‘3'l-mAbFB5, 5-Ri daily X 5, or 5-FU on days 1
and 5. Additional groups were treated either with a combination regimen of
5-nj
@
@
daily
X 5 +
‘311-mAb A33,
5-Ri
on days
I and 5 +
‘3'I-mAb
A33,
5-Ri daily X 5 + ‘3'I-mAb
FB5, or 5-Ri on days 1 and 5 + ‘311-mAb
FB5.
Growth controls consisted of mice receiving no treatment. In the second
experiment, the effectiveness of 5-FU + LV + ‘
‘I-mAb
A33 was compared
with that of ‘‘I-mAbA33 alone and with 5-FU + LV. Again, an untreated
control group was included. In the third experiment, the effect of 5-FU + LV
on tumor growth was compared to doxorubicin or carmustine, each with or
without ‘311-mAb
A33.
Statistical
Methods.
Within each treatment group, the growth curves of
tumors were fitted with a linear regression model, up to second order, of time
(day) after logarithmic transformation. The model can be displayed as:
log(tumor volume) = A + B X day + C X day2
The regression coefficients A, B, and C were estimated using a generalized
estimation
equation (30). In comparing
tumor growth between groups, the
AUC (measuring tumor volume accumulated over time) from day 0 to day 28
was the endpoint of comparisons, with AUC defined as:
B
AUC
=
28A
+
282_
C
+
2
28@—
3
where A, B, and C are the estimated regression coefficients.
A smaller AUC compared to the control group AUC indicates a treatment
effect on tumor growth inhibition. We assumed that the effect of a regimen
composed of two agents is equivalent to the sum of the separate effects of the
two agents plus the synergistic effect, if it exists. As an analogue to the test of
interaction in the two-way ANOVA, the synergy between agents A and B was
tested by applying a Z test on the difference between (a) AUC of treatment A
and B + AUC of control and (b) AUC of treatment A + AUC of treatment B.
If the tests of synergy were not significant, the tests of main effects were
pooled over different treatment combinations using the Z test. For observations
beyond 28 days, the proportion
of long-term survival between groups was
compared using Fisher's exact test. Three separate experiments were per
formed. Only the treatment groups within the same experiment were compared
to each other. For the comparisons within an experiment, Type I errors were
adjusted using Bonferroni's method.
RESULTS
A dose-response curve for ‘‘I-mAbA33 was established previ
ously4; the lowest dose of ‘311-m.Ab
A33 giving an antitumor effect
was 0.1 mCi. This dose was tested again and confirmed to be the
lowest dose producing the smallest measurable tumor-suppressive
effect. It was therefore selected for our studies (see below).
The mean AUCs from day 0 to day 28 for each group are shown in
Table I. Many of the AUCs are negative due to logarithmic transfor
mation of small values (< 1). In a preliminary experiment, 5-Ri was
tested at doses ranging from 15 to 30 mg/kg daily X 5 or at 40—75
mg/kg on days I and 5 (data not shown), but even the maximum dose
given by daily injection or on days 1 and 5 achieved only a minimum
antitumor effect (Fig. 1).
In an earlier study, unlabeled mAb A33 had shown no demonstrable
effect on the growth of established tumors in this model.4 Likewise,
the isotype-matched control ‘311-m.Ab
FB5 showed no significant
effect on tumor growth when compared to untreated controls (Fig. 2).
The combination of 5-FU given daily X 5 or on days 1 and
5 + ‘311-mAb
FB5 at 0. 1 mCi showed no increased tumor-suppressive
effects compared to 5-FU alone (Fig. 3). The combination of 5-FU
and ‘311-mAb
A33 resulted in significantly higher antitumor activity
(Fig. 4). In the groups of mice that were treated either with 5-FU
daily X 5 + ‘31I-mAbA33orwith5-FUondays
1 and5 + ‘311-mAb
A33, one xenograft in both groups escaped the therapy relatively
early. Three of the eight mice in each group developed minimal tumor
growth 54—60days after treatment. The other four animals in each
group had no evidence of tumor growth until 80—84 days after
treatment, when one of the four mice in each group had a recurrence.
The remaining three animals (38%) in both groups had no recurrence
as late as 276 days following treatment. Thus, statistical analysis
showed that long-term, disease-free survival for both of these corn
4 E.
Barendswaard,
S.
Welt,
F.
Daghighian,
A.
Scott,
M.
Graham,
and
unpublished observations.
2182
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L.
J.
Old,
COMBINATIONRADIOIMMUNOTHERAPY
AND CHEMOTHERAPY
agents did not improve the antiturnor
compared to radiolabeled antibody alone
Two deaths unrelated to tumor growth
one animal of eight (12.5%) in the
2.5
2.0
Control
1.5
activity of 13‘I-mAbA33
(data not shown).
occurred during treatment:
group treated with 5-Hi
daily X 5 + ‘31I-mAb
PBS and one ofsix (16.5%) in the group treated
1.0
with doxorubicin + ‘311-mAb
FB5. Minor weight loss was observed
in several animals treated with ‘31I-mAbPBS and with doxorubicin.
No signs of toxicity were seen in animals treated with 5-Ri and LV
0.5
0.0
as single agents and with any combinationplus ‘311-mAb
A33.
048121620242832364044485256606468
DISCUSSION
ci;-.
2.5
2
2M
E
5-Ri 30 mg/kg daily x 5
11.5
>
Adjuvant chemotherapy, radiation, and immunotherapy
have
shown some usefulness in the treatment of primary colorectal carci
norna and its more advanced stages (3 1). However, agents with better
antitumor activity need to be identified if significant progress is to be
made.
Recent studies have shown a significant survival benefit (6, 32) for
patients treated with chemotherapy and external beam radiation after
curative surgery for rectal cancer. The underlying cause for this
phenomenon is thought to be radiosensitization. In vitro and in vivo
studies of the combined effects of 5-Hi and radiation (2 1, 33—37)
have suggested that any increase of effect over radiation alone is not
due to inhibition of repair of sublethal or potentially lethal damage.
The mode of interaction of the drug with radiation remains unclear. In
addition, the sequencing of 5-Hi and radiation treatments may be a
crucial parameter. It has been suggested that synergism could be
achieved only by 5-Hi infusions, with brief injection schedules yield
ing additive effects (35). Other investigators (21, 38) found only
additive antitumor effects without evidence for sequence dependence.
Nevertheless, clinical trials have suggested a greater than expected
1.0
5-
0
E
—
0.0
048121620242832364044485256606468
2.5
@
@@..##F:_F(J
75 mg/kg on days 1 and 5
@
0.0!
t
t
I I
048121620242832364044485256606468
I
local control rate with 5-Hi chemoradiation for both squamous cell
I
days after start of treatment
Fig. I. Effect of 5-FU on tumor growth. Control, n = 8; MTS, 0.375 cm3. 5-Ri daily,
n = 6; MTS, 0.344 cm3. 5-FU bolus, n
6; MTS, 0.369 cm3.
carcinomas and adenocarcinomas (39).
In colon cancer, external beam radiation has its limits for technical
reasons and because of toxicity to adjacent organs. Lacking these
drawbacks, radiolabeled antibodies might be an alternative to deliver
2.5
bination therapies was significantly different from that of 5-Hi alone
or ‘311-rnAb
A33 alone (Fisher's exact test, P < 0.05). A synergistic
antitumor effect is suggested when the groups are compared for
@
Control
survival over the entire observation period (276 days).
@
A similar set of experiments comparing the combination of
5-Hi + LV and the addition of ‘311-mAbA33 showed that chemotherapy and radioimmunotherapy have significantly more antitumor
effects (Fig. 5), whereas the addition of ‘31I-mAbFBS to 5-PU + LV
did not increase antitumor activity significanfly (Fig. 5).
To determine whether combination therapy had additive or synergistic effect on reduction of tumor size, differences in tumor volume
were analyzed. When the tumor weight of mice reached 2 g, the
@
animals
@
2.0
1,5
were sacrificed.
Therefore,
the analysis
was carried
,— 0.5
‘
E
-@
>
048121620242832364044485256606468
2.5
out only
2 0
until tumor volumes reached 2 cm3. No statistical differences were
observed in the analysis evaluating synergistic antitumor effects (by
1.5
1311-mAbFBS
tumor volume) for the combination of 5-Ri given daily X 5 or on
days 1 and 5 + ‘311-mAbA33 and either treatment alone for days
1—28.However, the two different treatment modalities had additive
antitumor activity.
To determine whether the enhanced tumor-suppressive effect of
5-Hi
+ ‘311-mAb A33
chemotherapeutic
with
or without
LV
might
be common
1.0
0.5
0.0
048121620242832364044485256606468
to
days after start of treatment
agents in general or specific to certain drugs, we
tested combinations of doxorubicin + ‘311-mAbA33 and carmustine + ‘311-mAb
A33. The addition of these two chemotherapeutic
Fig.2. Effectof control‘311-mAb
J2@5
on tumorgrowth.Control,n = 8; MTS,0.375
cm3.‘311-mAb
FB5.n = 6; MTS.0.345cm3.
2183
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COMBINATION RADIOIMMUNOTHERAPYAND CHEMOTHERAPY
radiation therapy in colon cancer. The realistic evaluation of radiola
2.5
beled antibodies as radiotherapeutic agents, however, depends on
2.0
accurate dosimetry calculations. This is key information for radioim
munotherapy trials in solid tumors that will require continuous mon
itoring of isotope levels in essential tissues for the length of time the
isotope is retained. Positron emission tomography analysis offers an
approach to obtaining this information.
Another important consideration is rnicrodosimetry, which takes
into account the architecture of the tumor and the distribution of
1.5
1311-mAbA33
1.0
0.5
0.0
.@
(%J
c'J
%@ Q@o r'@
m
//@
%@
@O
‘0
antibody. Our earlier studies with mAb A33 have shown that local
ization was specific when
@mTc@albunün
was compared to radiola
2.5
2.0
2.5
5-FU30 mg/kg daily
xS
1.5
2.0
1.0
1.5
5-Ri 30 mg/kg daily x S
0.5
1.0
0.0
0.5
@
@
0
4—I--I
e'J
‘.4
-@
@O
m
0
@e
‘0
C'J
i―.
‘@
‘0
‘0
N.
01
0.0
5-FU 30 mg/kg daily x 5
048121620242832364044485256606468
‘@—2.5
E
+
w
E
@
25
@
2.0
@
1.5
+ 1311-mAb
@
0.5
FB5
c@-..
A33
@/I!
1
..—.—.
S
0.0
1.0
@
1.5
1.0
5-Ri 30 mg/kg daily x 5
@
1311-mAb
I
‘@ 2.0
0
-GO-C
(‘.4
‘-I
‘4
10
(,@
‘@
0
C―J
‘0
N.
‘0
10
01
N.
0.5
,@0.0
E
2.5
0 4 8121620242832364044485256606468
E
2.0
1.5
0
>
@
—
@
@
2.5
1.0
2.0
0.5
5-FU75mg/kgondays1and5
.
0.0
1.5
days I and S
0
c@4
—
‘4
c@J
‘0
0
(@J
—
‘a
r'j
‘0
rn
.
.
.
I,
-a
0
‘0
(Si
N-
@0
0
‘0
C@J
N-
‘4
‘0
01
F,
‘0
N.
1.0
0.5
2.5
0.0
2.0
048121620242832364044485256606468
1.5
1.0
@
@
2.5
0.5
2.0
0.0
1.5
5-nj75 mg/kgon
1.0
days 1 and 5 + 1311-mAbFB5
‘0
Q,
‘0
N.
days after start of treatment
Fig. 4. Effect of addition of specific 31I-mAb A33 radioimmunotherapy to chemo
therapy on tumor growth. ‘31I-mAb
A33, n = 8; MTS, 0.329 cm3. 5-RI daily, n = 6;
MTS, 0.344 cm3. 5-FU daily + ‘311-mAbA33, n = 8; MTS, 0.392 cm3. 5-Ri bolus,
n = 6; MTS, 0.369 cm3. 5-PU bolus 4- ‘311-mAb
A33, n = 8; MTS, 0.376 cm3.
0.5
0.0
0 4 8 121620242832364044485256606468
days after start of treatment
Fig. 3. Effect of nonspecific radioimmunotherapy
beled mAb A33 as a marker for tumor blood pool uptake, and when
in combination with chemotherapy
on tumor growth. 5-FU daily, n = 6; MTS, 0.344 cm3. 5-FU daily + ‘31I-mAb
FB5,
n = 8; MTS, 0.342 cm3. 5-FU bolus, n
FB5, n = 8; MTS, 0.349 cm3.
6; MTS, 0.369 cm3. 5-FU bolus + ‘311-mAb
a ‘31I-labeledisotype-matched control antibody was compared to
‘25I@@@b
A33 (28). Using autoradiographs, we were able to show that
isotope accumulation in tumors corresponded to the antibody binding
2184
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COMBINATION RADIOIMMUNOTHERAPYAND CHEMOTHERAPY
in other therapeutic systems, it was shown that human xenografts in
mice seem to retain histology and chemosensitivity of the original
tumor (40) and that radiosensitivity of individual tumor cells corre
lates well with the original tumor (41).
For the studies presented here, we selected a ‘311-mAbA33 dose
that was 20% of the MTD. On the basis of the % ID/g tumor observed
in this animal model, peak ‘@I
uptake corresponds to 0.035 mCi/g. In
our clinical studies with ‘311-mAb
A33, uptake (% Dig) was meas
ured by biopsy on day 7 or 8 after antibody administration. In
dosimetry studies, patients with the highest uptake of 1311-mAb A33
would achieve isotope concentrations of 0.015—0.045 mCi/g if treated
at the MTh (75 mCi/rn2). Thus, the effects observed in the present
2.5
2.0
5-FU+ leucovorin
1.5
1.0
0.5
0.0
048121620242832364044485256606468
‘@—
E
2
2.5
study may be correlated directly to the subgroup of patients with the
2.0
highest radiolocalization indices for ‘311-mAb
A33. Our results show
an additive effect of combination
chemotherapy
(5-Hi
or
5-Hi + LV) plus low-dose radioimmunotherapy. When survival was
used as an end point, a synergistic effect was found, in contrast to
earlier findings in a mouse radioimmunotherapy
model using an
anti-CEA antibody (42). No differences were observed in antitumor
11.5
>
1.0
+ leucovorin
5-
+ 1311-mAb
FB5
0
E 0.5
—
0.0
effects when the two 5-Hi treatment schedules were compared, and
048121620242832364044485256606468
2.5
2.0
1.5
S-nj + teucovorin
4
1311-mAb
A33
1.0
0.5
0.0
4 8 12 16 20 24 28 32 36 40 44 48 52 56 60 64 68
days after start of treatment
Fig. 5. Effect of addition of LV to combination chemoradioimmunotherapy.
5-FUlLy,
n = 8; MTS, 0.350 cm3. 5-FUlLy + ‘311-mAbFBS, n = 8; MTS, 0.325 cm3. 5-Ri!
LV + ‘31mAb
A33, n = 8; MTS, 0.387 cm3.
specifically to the tumor cells while the surrounding stromal cells and
vasculature did not concentrate the isotope. Thus, dosimetry calcula
tions have provided only estimates of radiation doses delivered to
tumor cells. Single-cell dosimetry with long-range @3-emitterssuch as
‘@‘i
provide an even greater challenge. In general, our present knowl
edge is based on external and biopsy-based measurements; however,
tumor tissue doses reported to be absorbed are only a fraction of what
is needed to eradicate tumors (27).
Human cancer xenograft models in nude mice have proved to be
valuable in testing the therapeutic potential of new reagents such as
radiolabeled antibodies. However, when evaluating the results of
these models, an important consideration is the limitation in predicting
efficacy in human studies. One major consideration is that the anti
body uptake by the tumor can be 3000-fold higher in the mouse model
than in patient studies (30% versus 0.01% DIg tumor). This is
partially compensated for by the higher total dose used in humans
compared to mice. Because the MTD of 131!in humans is almost 300
times that in mice, this still leaves a 10-fold higher tumor tissue dose
in mice than in humans at the MTD. Thus, it is important to select
doses for experiments with animal models that are a fraction of the
MTD. In general, animal models may in some cases exaggerate the
antitumor effect of experimental therapy regimens, as it does with
radiolabeled antibodies. However, when this issue has been examined
both demonstrated enhanced activity when combined with specific
radioimmunotherapy. In the clinic, the dose-limiting toxicity for bolus
and continuous-infusion 5-Ri is different, and the relative lack of
hematological toxicity of the continuous-infusion schedule makes this
an attractive partner for ‘311-labeled
antibody-based therapies. Due to
the low nontumor radiation dose and specific uptake of radiolabeled
antibody, we could not detect any signs of toxicity in our animals. In
patients treated with 5-Hi/mAb A33 chemoradioimmunotherapy,
we
might expect enhanced antitumor effects with little change in toxicity,
due to the low nonspecific radiation doses delivered to critical tissues
manifesting 5-Ri toxicity. The enhanced antitumor effects were spe
cific for 5-Hi-based treatments as the addition of other chemothera
peutic agents (doxorubicin and carmustine or even LV) to ‘311-mAb
A33 had no beneficial effect.
In theoty, mAbs can deliver radiation therapy continuously over pro
longed periods. Hyperfractionated radiation dosing, in which the rationale
is based on growth and on radiation repair patterns of tumor cells, has
shown advantages over single doses (43—45).After a single antibody
administration, the actual dose rate to the tumor does not simulate
hyperfractionation protocols due to the half-life effect of the isotope used
(e.g., ‘@‘I)
and the continuous clearance of isotope from the tumor. With
the generation and clinical use of humanized antibodies, therapeutic
regimens may be developed that will approximate hyperfractionation
because the humanized antibody can be given repeatedly.
The question of whether the response to continuous, exponentially
decreasing irradiation, which is characteristic of radioimmunotherapy,
is biologically equivalent to conventionally fractionated, high-dose
rate irradiation remains to be solved. Very few studies have addressed
this question (46—53).Our results here have clearly demonstrated an
improvement in antitumor effects by chemoradioimmunotherapy in an
animal model. This finding should be tested in clinical studies in
subsets of patients with advanced disease, who have not yet been
treated with 5-Hi. We have observed that in a group of heavily
pretreated patients, who received chemotherapy after a Phase I radio
immunotherapy trial with ‘251-mAbA33, a significant number had
major responses (54). These unexpected findings need to be explored
further and verified in well-designed clinical studies. Preclinical stud
ies to identify other chemotherapeutic agents with additive or syner
gistic activity when combined with radiolabeled antibodies should
continue, especially with the newer agents identified recently as
having activity in colon cancer.
2185
Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1997 American Association for Cancer Research.
COMBINATION RADIOIMMUNOTHERAPYAND CHEMOTHERAPY
29. Houghton, P. J., Houghton, J. A., Myers, L, Chesire, P., Howbert, J. J., and Grindey,
REFERENCES
G. B. Evaluationof N-(5-indanylsulfonyl)-N-(4-chlorophenyl)-urea
againstxenograftsof
I . Schneebaum, S., and Arnold, M. W. Adjuvant treatment for rectal cancer: current
status. Oncology (Basel), 7: 83—90,1993.
2. Gastrointestinal Tumor Study Group. Prolongation of the disease free interval in
surgically treated rectal carcinoma. N. EngI. J. Med., 312: 1465—1472,
1985.
3. Douglass, H. 0., Jr., Moertel, C. G., Mayer, R. J., Thomas, P. R., Lindblad, A. S.,
Mittleman, A., Stablein, D. M., and Bruckner, H. W. Survival after postoperative
combination treatment of rectal cancer. N. EngI. J. Med.. 315: 1294—1295, 1986.
4. Fisher, B., Wolmark, N., Rockette, H., Redmond, C., Deutsch, M., Wickerham, D. L.,
Fisher, E. R., Caplan, R., Jones, J., and Lamer, H. Postoperative adjuvant chemo
therapy or radiation therapy for rectal cancer: results from NSABP protocol R-Ol.
J. NatI. Cancer Inst., 80: 21—28,1988.
5. Gastrointestinal
Tumor Study Group. Radiation therapy and fluorouracil
with or
without semustine for the treatment of patients with surgical adjuvant adenocarci
noma of the rectum. J. Clin. Oncol., 10: 549—557,1992.
6. Krook, J. E., Moertel, C. G., Gunderson, L. L., Wieand, H. S., Collins, R. T., Beart,
U. R. W., Kubista, T. P., Poon, M. A., Meyers, W. C., Mailliard, J. A., Twito, D. I.,
Morton, R. F., Veeder, M. H., Witzig, T. E., Cbs, S., and Vidyarthi, S. C. Effective
surgical adjuvant therapy for high-risk rectal carcinoma. N. Engl. J. Med., 324:
709—715,
1991.
7. Comis, R. L., and Carter, S. K. Integration of chemotherapy into combined modality
treatment of solid tumors. Cancer Treat. Rev., I: 221—238,1974.
8. Ramming. K. P., and Haskell, C. M. Colorectal malignancies. In: C. M. Haskell (ed),
Cancer Treatment, Ed. 2., pp. 295—334.
Philadelphia: W. B. Saunders, 1985.
9. Cohen, A. M., Shank, B., and Friedman, M. A. Colorectal cancer. In: V. T. De Vita,
Jr., S. Hellman, and S. A. Rosenberg
(eds.), Cancer: Principles and Practice of
Oncology, Vol. 1, Ed. 3, pp. 895—964.
Philadelphia: J. B. Lippincott, 1989.
10. Bleyer, W. A. New vistas for leucovonn in cancer chemotherapy. Cancer (Phila.), 63:
995—1007, 1989.
I 1. Rustum, Y. M. Toxicity and antitumor activity of 5-fluorouracil in combination with
leucovorin.
Cancer(Phila.).
63: 1013-1017,1989.
12. Houghton, J. A., Williams, L. G., Loftin, S. K., Cheshire, P. J., Morton, C. L.,
Houghton, P. J., Dayan. A., and Jolivet, J. Factors that influence the therapeutic
activityof 5-fluorouracil(6RS)leucovorincombinationsin colonadenocarcinoma
xenografts. Cancer Chemother. Pharmacol., 30: 423—432,1992.
I3. Advanced Colorectal Cancer Mets-analysis Project. Modulation of fluorouracil by
rates. J. Clin. Oncol., 10: 896—903,1992.
14. Laufman, L. R., Brenckman, W. D., Stydnicki, K. A., Morgan, E. D., Collier, M.,
Knick, V. B., Mullin, R., and Ferone, R. Clinical experience with leucovorin and
5-fluorouracil. Cancer (Phila.), 63: 1031—1035,1989.
15. Arbuck, S. G. Overview of clinical trials using 5-fluorouracil and leucovorin for the
treatmentof colorectalcancer.Cancer(Phila.),63: 1036—1044,
1989.
16. Mini, E., Trave, F., Rustum, Y. M., and Bertino, J. R. Enhancement of the antitumor
effects of 5-fluorouracil by folinic acid. Pharmacol. Ther., 47: 1—19,
1990.
17. Intemational Multicenter Pooled Analysis of Colon Cancer Trials (IMPACT) Inves
tigators. Efficacy of adjuvant fluorouracil and folinic acid in colon cancer. Lancet,
345: 939—944,1995.
18. Laufman, L. R., Bukowski, R. M., Collier, M. A., Sullivan, B. A., McKinnis, R. A.,
Clendennin, N. J., Guaspari, A., and Brenckman, W. D., Jr. A randomized, double
plus placebo versus fluorouracil plus oral leucovorin in
patients with metastatic colorectal cancer. J. Clin. Oncol., II: 1888—1893,1993.
19. Leichman, C. G., Fleming. T. R.. Muggia, F. M., Tangen, C. M., Ardalan, B.,
Doroshow, J. H., Meyers, F. J., Holcombe, R. F., Weiss, G. R., and Mangalik, A.
Phase II study of fluorouracil and its modulation in advanced colorectal cancer: a
Southwest Oncology Group Study. J. Clin. Oncol., 13: 1303—1
3 11, 1995.
20. Fu, K. F., Rayner, P. A., and Lam, K. N. Modification of effects of continuous low
dose rate irradiation by concurrent chemotherapy infusion. Int. J. Radiat. Oncol. Biol.
Phys.,10: 1473—1478,
1984.
21 . Weinberg, M. J., and Rauth, A. M. 5-Fluorouracil infusions and fractionated doses of
radiation: studies with a murine squamous cell carcinoma. mt. J. Radiat. Oncol. BioI.
Phys.,13: 1691—1699,
1987.
22. Byfield, J. E., Chan, P. Y. M., and Seagren, S. L. Radiosensitization
18: 74, 1977.
23. Byfield, J. E., Barone, R. M., Seagren, S. L., Frankel, S., Quinol, L., and Mendelsohn,
the survival of mammalian cells in vitro and on early recovery between fractionated
X-ray doses. Br. J. Radiol., 34: 458—465,1966.
35. Byfield, J. E., Calabro-Jones, P., Klisak, I., and Kullianian, F. Pharmacologic require
mentsfor obtainingsensitizationof humantumorcellsin vitroto combined5-flu
orouracil or Ftorafur and X-rays. tnt. J. Radiat. Oncol. Biol. Phys., 8: 1923-1933,
1982.
36. Nakajima, V., Miyamoto, Y., Tanabe, M., Watanabe, I., and Terasima, T. Enhance
ment of mammalian cell killing by 5-fluorouracil in combination with X-rays. Cancer
Res., 39: 3763—3767,1979.
37. Rich, T. A., Kavanaugh, B., Williams, M., Bock, S., Murray. D., Meyn, R., and
Brock, W. A. Effects of continuous post-irradiation low dose 5-fluorouracil on log
and plateau phase CHO cells. Proceedings, Chemical Modifiers ofCancer Treatment,
Clearwater, FL, Abstr. 6—21,1985.
38. Stephens, J. C., Peacock, S. H., and Steel, G. G. Cell survival in Bl6 melanoma after
treatment with combination of cytotoxic agents: lack of potentiation. Br. J. Cancer,
36: 84—93,1977.
39. Schein, P. S., Gore, M. E., and Buckman, R. A. Radiation therapy and 5-fluorouracil.
Drugs Exp. Clin. Rca., 8: 587—592,1982.
40. Winograd, B., Boven, E., Lobezzo, M. W., and Pinedo, H. M. Human tumor
xenografts in the nude mouse and their value as test models in anti-cancer drug
development. In Vivo, 1: 1—14,
1987.
growth delay and survival data. in Vivo,4: 253—258,1990.
42. Chalandon, Y., Mach, J-P., Pèlegrin, A., Folli, S., and Buchegger, F. Combined
radioimmunotherapy and chemotherapy of human colon carcinoma grafted in nude
mice: advantages and limitations. Anticancer Res., 12: 1131—1140,1992.
43. Fowler, J. F. Briefsummary ofradiobiological principles in fractionated radiotherapy.
Semin. Radiat. Oncol., 2: 16—21,1992.
44. Fowler, J. F. Intercomparisons of new and old schedules in fractionated radiotherapy.
Semin. Radiat. Oncol., 2: 67—72,1992.
45. Thames, H. D., Jr. Withers, H. R., and Fletcher, G. H. Changes in early and late
radiation responses with altered dose fractionation: implications for dose survival
relationship. Int. J. Radiat. Oncol. Biol. Phys., 8: 219—226, 1992.
46. Knox, S. J., Goris, M. L., and Wessels, B. W. Overview of animal studies comparing
radioimmunotherapy with dose equivalent external beam irradiation. Radiother. On
col.,
23: 111—117,
1992.
47. Buchsbaum, D. J., Ten Haken, R. K., Heidorn, D. B., Lawrence, T. S., Glatfelter,
A. A., Terry, V. H., Guilbault, D. M., Steplewski, Z., and Lichter, A. S. A comparison
of ‘311-labeledmonoclonal antibody 17—IAtreatment to external beam radiation on
the growth of LSI74T human colon cancer xenografts. mt. J. Radiat. Oncol. Biol.
Phys., 18: 1033-1041, 1990.
48. Buras, R. R., Wong, J. Y. C., and Kuhn, J. A. Comparison of radioimmunotherapy
and external beam radiotherapy in colon cancer. Int. J. Radiat. Oncol. Biol. Phys., 16:
12—16,1991.
49. Knox, S. J., Levy, R., Miller, R. A., Uhland, W., Schiele, J., Ruehl, W., Finston, R.,
monoclonal antibodies. Cancer Res., 50: 4935—4940, 1990.
50. Neacy, W. P., Wessels, B. W., Bradley, E. W., Kovandi, S., Justice, T., Danskin,
S., and Sands, H. Comparison of radioimmunotherapy and 4 MV external beam
J. Biweekly infused 5-fluorouracil and X-rays in advanced gastro-intestinal cancer.
Proc. Am. Soc. Clin. Oncol., 18: 322—324,1978.
24. Calabro-Jones, P. M., Byfield, J. E., Ward, J. F., and Sharp, T. R. Time dose
relationships for 5-fluorouracil cytotoxicity against human epithelial cells in vitro.
radiotherapy of tumor xenografts in athymic mice. J. Nucl. Med., 27: 902—903,
1986.
51 . Palme, D. F., Berkopec, J. M., Elson, M. K., Bono, M. D., Wessels, B. W., Lange,
P. H., andVessella, R. L. Immunotherapyof renalcell carcinomaxenograftsby 1-131
monoclonal antibody A6H compared to single fraction external beam irradiation.
Cancer Res., 42: 4413—4415,1982.
F., and Valeriote, F. Combined effect of X-radiation
and
5-fluorouracil on survival of transplanted leukemia cells. J. NatI. Cancer Inst., 47:
865—870,
1971.
26. Welt, S., Divgi, C. R., Kemeny, N., Finn, R. D., Scott, A. M., Graham, M., St.
Germain, J., Carswell Richards, E., Larson, S. M., Oettgen, H. F., and Old, L. J. Phase
I/Il study of iodine 131-labeled monoclonal antibody A33 in patients with advanced
colon cancer. J. Clin. Oncol., 12: 1561—1571,
1994.
J. Nucl. Med., 28: 651, 1987.
52. Vessella, R. L., Palme, D. F., Berkopec, J. M., Elson, M. K., Truillo, G., Wessels,
B. W., andLange,P. H. A comparisonof escalatingsingle andmultiplefractionlow
dose rate radioimmunotherapy with monoclonal antibody A6H 1-131 conjugates to
high dose rate external beam therapy on human renal cell carcinoma xenografts.
J. Nucl. Med., 29: 875, 1988.
53. Wessels, B. W., Vessella, R. L., Palme, D. F., Berkopec, J. M., Smith, G. K., and
Bradley, E. W. Radiobiological comparison of extemal beam irradiation and radio
27. Goldenberg, D. M. (ed). Cancer Therapy with Radiolabeled Antibodies. Boca Raton:
CRC Press, 1995.
28. Welt, S., Divgi, C. R., Real, F. X., Yeh, S. D., Gain
Lancet, 343: 1177—1
183, 1994.
32. Gastrointestinal Tumor Study Group. Survival after postoperative combination treat
ment of rectal cancer. N. Engl. J. Med., 315: 1294—1295,1986.
33. Bagshaw, M. A. Possible role of potentiators in radiation therapy. Am. J. Roentgenol.,
85: 822—833,1961.
34. Berry, R. J. Effects of some metabolic inhibitors on X-ray dose response curves for
Day-Lollim, P., and Goris, M. L. Determinants of the antitumor effect of radiolabeled
by 5-Ri:
molecular origins and clinical scheduling implications. Proc. Am. Assoc. Cancer Res.,
25. Vieni, T., Eggerding,
Wine, J., and German Cancer Aid 17—IAStudy Group. Randomised trial of mono
clonal antibody for adjuvant therapy of resected Dukes C colorectal carcinoma.
41. Schwachofer, J. H. M., Hoogenhout, J., Kal, B., Koedam, J., and Van Wezel, H. P. N.
Radiosensitivity of different human tumor lines grown as xenografts determined from
leucovorin in patients with advanced colorectal cancer: evidence in terms of response
blind trial of fluorouracil
pediatric rhabdomyosarcoma. Cancer Chemother. Pharmacol., 24: 84—88,1989.
30. Liang, K., and Zeger, S. L. Longitudinal data analysis using generalized linear
models. Biometrika, 73: 13—22,1986.
31. RiethmUller, G., Schneider-GEdicke, E., Schlimok, G., Schmiegel, W., Raab, R.,
Hdffken, K., Gruber, R., Pichlmaier, H., Hirche, H., Pichimayr, R., Buggisch, P.,
Chesa, P., Finstad, C. L.,
Sakamoto, J., Cohen, A., Sigurdson, E. R., Kemeny, N., Carswell, E. A., Oettgen,
H. F., and Old, L. L. Quantitative analysis of antibody localization in human
metastatic colon cancer: a phase I study with monoclonal antibody A33. J. Clin.
Oncol., 8: 1894—1906,1990.
immunotherapy in renal cell carcinoma xenografts. mt. J. Radiat. Oncol. Biol. Phys.,
17: 1257—1263,
1989.
54. Welt, S., Scott, A. M., Divgi, C. R., Kemeny, N. E., Finn, R. D., Daghighian, F., St.
Germain, 1., Richards, E. C., Larson, S. M., and Old, L J. Phase 1/11study of iodine
125-labeled monoclonal antibody A33 in patients with advanced colon cancer. J. Clin.
Oncol., 14: 1787—1797,
1996.
2186
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Enhanced Antitumor Activity of Combination
Radioimmunotherapy ( 131I-labeled Monoclonal Antibody A33)
with Chemotherapy (Fluorouracil)
Jörg Tschmelitsch, Els Barendswaard, Clarence Williams, Jr., et al.
Cancer Res 1997;57:2181-2186.
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