Download Therapeutic Effect of Treatment with Polyclonal

[CANCER RESEARCH 47, 4293-4295, August 15, 1987]
Therapeutic Effect of Treatment with Polyclonal or Monoclonal Antibodies to aFetoprotein That Have Been Conjugated to Daunomycin via a Dextran Bridge:
Studies with an a-Fetoprotein-producing
Rat Hepatoma Tumor Model1
Yutaka Tsukada,2 Kiyoshi Ohkawa, and Nozomu Hibi
Department of Biochemistry, School of Medicine, Hokkaido University, Sapporo, Japan (Y. T.J; Department of Microbiology, Sapporo Medical College, Sapporo, Japan
[K. O.J; and Tumor Laboratory, Kokubunji, Tokyo, Japan [N. H.]
ABSTRACT
Purified monoclonal mouse or polyclonal horse antibodies (Ab) to rat
a-fetoprotein (AFP) were conjugated with daunomycin via a dextran
bridge. The therapeutic effect of these Ab-daunomycin conjugates on an
AFP-producing rat hepatoma which was inoculated s.c. in Donryu rats
was studied. Tumors (s.c.) and distant métastaseswere present by 14
days after tumor inoculation and the serum AFP level was 35 nv,lm\. The
injection of the Ab-daunomycin conjugate, started on day 14, significantly
prolonged host survival with inoculated controls having a median survival
of 25 days compared to 57 and 60 days for the treated groups. In a
second study the Ab-daunomycin conjugates were injected i.v. every other
day for five times after the surgical resection of the s.c. tumor. There
was a slight therapeutic effect with either antibody or daunomycin alone
but treatment with the AFP Ab-daunomycin conjugates significantly
prolonged survival and 60% of these treated animals were "tumor free"
when sacrificed on day 100. Serial quantitation of the concentration of
AFP in the serum of the treated tumor-bearing or in the tumor-resected
rats correlated with the therapeutic effectiveness of the Ab-daunomycin
conjugates. These experiments show that the optimal treatment with
specific antibody-drug conjugates will be in hosts where there is a small
residual tumor burden such as may exist following resection of a primary
tumor mass. They further show that the serial quantitation of serum AFP
can be utilized to determine if residual tumor is present following treat
ment with Ab-daunomycin conjugates.
cells into the right thigh. By day 14 solid tumors which were 15-20
mm in diameter were present and microscopic examination of sacrificed
animals showed that all of them had micrometastases in their lungs
(5). The serum AFP averaged 35 Mg/ml (6).
Statistical Analysis. For statistical analysis Student's i test was used.
Antibody-Drug Conjugate. Purified polyclonal and monoclonal anti
bodies to rat AFP were conjugated to the antineoplastic drug dauno
mycin via a dextran (Dextran T-10; Pharmacia, Sweden) bridge as
previously described (7-10). The conjugate retained both the antibody
and drug activity. The molar ratio of antibody to daunomycin was 1 to
50 (3, 4). Various controls were prepared in a similar fashion (10).
AFP Determination. Serum AFP level was determined by radioimmunoassay (11). Blood samples were taken from the tail vein.
Chemical Reagents. All of the chemical reagents used were of analyt
ical grade.
RESULTS
In the first study tumor-bearing rats were injected intrave
nously with 200 fig of daunomycin every other day for 5 times
with treatment being initiated on the 14th day after inoculation
of the tumor (Fig. 1). Nontreated control rats or rats treated
with normal horse immunoglobulin died from the progressive
tumor growth and they had a median survival of 25 days. Rats
treated with monoclonal or polyclonal antibodies to AFP sur
INTRODUCTION
vived slightly longer and they had a median survival time of 32
days. The median survival days of rats which received normal
It has been hoped that polyclonal and monoclonal antibodies
horse immunoglobulin-daunomycin
conjugate, daunomycin
against tumor-associated antigens could be conjugated to chemotherapeutic agents and that these antibody-drug conjugates
alone, or a mixture of the polyclonal antibody to AFP and
would increase the effectiveness of chemotherapeutic agents in daunomycin ranged from 40 to 48 days. The longest median
survival of 57 and 60 days, respectively, was observed in the
the treatment of cancer. We have previously reported that
specific antibodies to AFP3 inhibited tumor growth in vitro as group of rats which received either the monoclonal or the
polyclonal antibody to AFP-daunomycin conjugate.
well as in vivo (1,2) and that the specific antibody-drug conju
Serum AFP concentrations were quantitated in all of the
gates were more effective than either antibody or drug alone
(3). Further studies showed that affinity-purified polyclonal and animals just prior to their demise (Fig. 2). High serum AFP
monoclonal antibodies to AFP that were conjugated with antilevels of 200 ^g/rnl was observed in the nontreated group and
in the group of rats injected with normal horse immunoglobulin,
cancer drugs were highly effective in therapy (4). In this report
daunomycin, and conjugate of normal horse immunoglobulin
we have investigated the effect of AFP antibody daunomycin
conjugates on the survival of rats with a s.c. tumor or on the and daunomycin. Moderate elevations in the serum from 12 to
20 Mg/ml was observed in rats injected with either the monoclo
survival of rats following surgical resection of a s.c. tumor mass.
nal or the polyclonal antibodies to AFP or with the mixture of
We found that the most effective treatment with the antibody
polyclonal antibody to AFP and daunomycin. Low serum AFP
daunomycin conjugates occurred when treatment was initiated
levels from 2 to 3.8 Mg/ml was observed in the rats which were
after the surgical resection of the primary tumor.
injected with the conjugates of monoclonal or polyclonal anti
bodies and daunomycin.
MATERIALS AND METHODS
We next examined the effect of specific conjugate therapy
ExperimentalAnimaland Hepatoma Cells. Donryu rats weighing 200 after surgical resection of the s.c. tumor (Fig. 3). The resection
g were used in this experiment. Rats were inoculated s.c. with I x Id'1
of the tumor at day 14 after inoculation of AH66 cells prolonged
the lives of rats significantly (P < 0.05) compared to no treat
Received 12/5/86; revised 5/6/87; accepted 5/13/87.
The costs of publication of this article were defrayed in part by the payment
ment. The rats which were treated with monoclonal or poly
of page charges. This article must therefore be hereby marked advertisement in
clonal
AFP antibody, daunomycin, normal horse immunoglob
accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1This work was supported in part by a (.runt in Aid for Cancer Research from
ulin-daunomycin conjugate, or a mixture of polyclonal antibody
the Ministry of Education, Science and Culture, Japan.
to AFP and daunomycin, after surgical resection of primary
2To whom requests for reprints should be addressed, at Department of
tumor, showed a significant prolongation of survival when
Biochemistry, School of Medicine, Hokkaido University, Sapporo 060, Japan.
3The abbreviation used is: AFP, a-fetoprotein.
compared to only surgical resection or to surgical resection plus
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POSTOPERATIVE EFFECT OF ANTIBODY-DRUG
PoAb+DM
CONJUGATE
IOO
no
PoAb-0«-DM
Nord
RM»ct*nlfll ^
MoAb
oAb-D«-DM
90
ttttt2S50
75
Doyi
Fig. 1. Antitumor effect of monoclonal or polyclonal AFP antibody-daunomycin conjugate. One million (large arrow) of AH66 hepatoma cells were inocu
lated s.c. at the right thigh of Donryu rats. The tumor cells grew up to the solid
tumor of 15-20 mm in diameter with the serum AFP level of 35 Mg/ml. The
treatment was initiated 14 days after inoculation every other day for Pive doses
(small arrows). One dose included 2 mg of purified antibody combined with 200
lit, of daunomycin. Five rats were used for each group.
, nontreated group
(No Tr.);
, normal horse immunoglobulin 2 mg (nig); —O—,
monoclonal
antibody to rat AFP 2 mg (MoAb); —•—,
polyclonal antibody to rat AFP 2 mg
(PoAb); —A—,
conjugate of normal horse immunoglobulin (2 mg) and dauno
mycin (200 /ig) (nlg-Dex-DM); —G—,daunomycin 200 ng (DM); —A—,mixture
of polyclonal antibody to rat AFP (2 mg) and daunomycin (200 ng) (PoAb +
DM); --A--,
monoclonal antibody to rat AFP (2 mg) daunomycin (200 ng)
conjugate (MoAb-Dex-DM); —•—,
polyclonal antibody to rat AFP (2 mg) dau
nomycin (200 /ig) conjugate (PoAb-Dex-DM).
Fig. 3. Antitumor effect of monoclonal or polyclonal AFP antibody dauno
mycin conjugates after surgical resection of a s.c. tumor mass. One million (large
arrow) of AH66 hepatoma cells were inoculated subcutaneously at the right thigh
of Donryu rats. The tumor masses were excised on day 14 after inoculation. (4)
Antibody treatment was initiated that same day and every other day for 5 doses
(small arrows).
, nontreated group (No Tr.);
, resection (Resect.);
—D—,resection plus normal horse immunoglobulin (Resect + nig); —*—,
resection plus monoclonal antibody to rat AFP (MoAb); —O—,resection plus
polyclonal antibody to rat AFP (PoAb); —A—,resection plus conjugate of normal
horse immunoglobulin and daunomycin (nlg-Dex-DM); —A—,resection plus
daunomycin (DM); —•—,
resection plus mixture of polyclonal antibody to rat
AFP and daunomycin (PoAb + DM); - -A- -, resection plus monoclonal antibody
to rat AFP daunomycin conjugate (MoAb-Dex-DM); --•—, resection plus
polyclonal antibody to rat AFP daunomycin conjugate (PoAb-Dex-DM).
jug/ml
1000
I000>jg/ml
DM »Résection
limono
AFP Ab.-D««-DM
Fig. 2. Serum AFP level at terminal stage. Serum AFP level at terminal stage
of rats in Fig. I experiment was determined. n.Ho.IgG, normal horse immuno
globulin; DM, daunomycin; poly AFP Ab, polyclonal antibody to rat AFP; Mono
AFP Ab, monoclonal antibody to rat AFP; AFP Ab + DM, mixture of polyclonal
antibody to rat AFP and daunomycin; nHo-IgG-Dex-DM, conjugate of normal
horse immunoglobulin and daunomycin; poly AFP Ab-Dex-DM, polyclonal anti
body to rat AFP daunomycin conjugate; mono AFP Ab-Dex-DM, monoclonal
antibody to rat AFP daunomycin conjugate.
Ab.-Dex-DM «-Resection
administration of normal horse immunoglobulin (P < 0.05).
There was a striking therapeutic effect of treatment of animals
with the monoclonal or polyclonal AFP antibody-daunomycin
t
20
30
conjugates in that three of five rats in each group (60%) survived
ÕÕTïï
until 100 days after inoculation of tumor. These animals were
Fig. 4. Change in serum AFP level. Serum AFP level of Fig. 3 experiment
sacrificed at that time and were found to be tumor free.
during the experimental days was plotted in each group. The blood was collected
from the tail vein. For simplicity, serum AFP level of rats received the resection
The serum AFP concentrations were determined in these
plus conjugate of normal horse immunoglobulin and daunomycin and rats re
resected animals during the experimental period (Fig. 4). Fol
ceived the resection plus mixture of polyclonal antibody to rat AFP and dauno
lowing resection of the primary solid tumor there was an mycin was omitted. Ab includes monoclonal and polyclonal antibodies. Symbols
are same as Fig. 1.
immediate decrease of the serum AFP level and this was fol
lowed by a rapid increase. This increase was due chiefly to the
production of AFP by the proliferating residual hepatoma cells. body treatment markedly suppressed serum AFP levels after
The groups which were treated with daunomycin showed a little
surgical resection of the tumor. A similar but greater suppres
delay in the second AFP peak. Monoclonal or polyclonal antision of the serum AFP occurred in the rats which were injected
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POSTOPERATIVE
EFFECT OF ANTIBODY-DRUG
with monoclonal or polyclonal AFP antibody-daunomycin con
jugate. It is of note that in those animals which were judged to
be tumor free by day 100 there were extremely low levels of
AFP at 10ng/ml.
CONJUGATE
REFERENCES
DISCUSSION
Specific antiserum to AFP has an inhibitory effect of the
growth of AFP-producing tumor cells (1). This has been con
firmed by Mizejewski et al. using mouse hepatoma ceil lines
(12, 13). Affinity-purified monoclonal and polyclonal antibod
ies to AFP have been utilized and proved to be equally effective
as whole antiserum in inhibiting the growth of tumor cells (4).
Conjugation methods were developed which made it possible
to use intermediate macromolecules as carriers so that anticancer drugs could be conjugated to affinity-purified antibodies
(14, 15-19). Successful therapeutic efforts have been made to
target chemotherapy by utilizing conjugating purified antibod
ies with chemotherapeutic agents in rat and mouse systems (3,
4, 14) and more recently with human tumor cells lines trans
planted in nude mice (20, 21).
In the experiments described in this paper we have used the
conjugate of specific antibodies with daunomycin via a dextran
bridge and studied what effect this treatment has on a solid
tumor model which has micrometastasis. The administration
of either monoclonal or polyclonal antibody to AFP-daunomycin conjugates to animals having a s.c. tumor significantly
prolonged survival when compared to the polyclonal antibody
to AFP and daunomycin. The median survival time of rats
receiving the conjugate was 2-fold that of the nontreated group.
In a second study, groups of rats were first treated with surgical
resection and then treated with various combinations of anti
body or antibody drug conjugates. Six of 10 rats injected with
the monoclonal or polyclonal antibodies conjugated with dau
nomycin were "cured" of their residual micrometastatic disease
as they were found to be microscopically tumor free at day 100.
Since we had previously shown that 100% of the animals at 14
days had microscopic métastases
in their lungs (5), these exper
iments mean that the remaining small foci of lung métastases
were effectively killed by the antibody drug treatment. This
latter study shows that the optimal time for antibody drug
conjugate treatment may be immediately postoperatively when
there is minimal residual tumor burden. Serum studies in which
the concentration of AFP was quantitated showed that the
optimal therapeutic effect was obtained in those animals treated
with the antibody-daunomycin conjugates.
ACKNOWLEDGMENTS
We acknowledge the skilled technical assistance of Miyuki Kitamura
as well as Masae Takada in typing this manuscript.
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Downloaded from cancerres.aacrjournals.org on August 3, 2017. © 1987 American Association for Cancer Research.
Therapeutic Effect of Treatment with Polyclonal or Monoclonal
Antibodies to α-Fetoprotein That Have Been Conjugated to
Daunomycin via a Dextran Bridge: Studies with an α
-Fetoprotein-producing Rat Hepatoma Tumor Model
Yutaka Tsukada, Kiyoshi Ohkawa and Nozomu Hibi
Cancer Res 1987;47:4293-4295.
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