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PREVENTIVE
EFFECTS OF A MONOCLONAL
ANTI-IDIOTYPIC ANTIBODY COMPARED
TO A HORMETIC MODEL ON RAT SARCOMAS
Preventive
effects of an antibody on rat sarcomas
S. FAIDERBE,
J.L. CHAGNAUD, M. BASTIDE*, P. DORFMANN**
and M. GEFFARD
Laboratoire PIOM, ENSCPB, BP 108, Avenue Pey
Berland , 33402 Talence cedex, France,
* Laboratoire d'Immunologie,
Faculté de Pharmacie, Université de Montpellier I, 15 avenuevCharles Flahault, 34060
Montpellier cedex 1, France
2 - inexistent
3 - Material and Methods
4 - Results
5 - Discussion
Increased levels of autoAb have previously been found in patients with malignant tumors (Chagnaud et al., 1992 ; Faiderbe et al., 1990; 1991; 1992a). These autoAb reflect antigenic disturbances between endogenous B(a)P ligand and its cytosolic receptor(s), and disturbances in PtdIns turn over. In the same time, a chemically-induced malignant tumor model was obtained after subcutaneously (s.c.) administration of a single dose of B(a)P (0.1mg/g body weight at the top of the right thigh of SD female rats) diluted in sesame oil (Faiderbe et al., 1992b). Significantly elevated levels of circulating anti-PtdIns autoAb were found in sera of B(a)P-treated rats 40 days after B(a)P administration, whereas no significant levels of anti-PtdIns autoAb were noted in control rats (oil-treated rats) (Faiderbe et al., 1992b). After day 60, autoAb levels plateaued in B(a)P-treated rats, and highly malignant sarcomas appeared with 100% efficiency around day 100. When a monoclonal anti-idiotypic Ab, internal image of conjugated B(a)P, called AIB1 Ab (Chagnaud et al., 1993a), was injected into SD female rats to mimic carcinogenic B(a)P effects, significant anti-PtdIns autoAb levels appeared. These circulating autoAb were found immunochemically equivalent to that found after injection of 2mg B(a)P (Chagnaud et al., 1993b). These results suggested possible relationships between carcinogen ligand/receptor interactions and PtdIns turn over.
AIB1 Ab effects on rat sarcomas have been evaluated before (day 73) and after (day 102) the appearance of clinically palpable tumors (Chagnaud et al., 1993c ; Chagnaud et al., 1994).We have found that when AIB1 Ab was administered to SD female rats, anti-PtdIns autoAb levels were modified (Chagnaud et al., 1993b, 1994). Furthermore, we have demonstrated that AIB1 Ab has the capacity to delay tumor onset, slow down growth, and to increase animal survival when administered biweekly to SD female rats, at different time after tumor induction (Chagnaud et al., 1993c, 1994).
The present investigation was designed to evaluate the possible vaccine effects of AIB1 Ab. Taking into account our previous results, we hypothesize that this anti-idiotypic Ab might have preventive effects when administered prior to carcinogen injection (tumor induction). To support this hypothesis, we have studied the effects of 500 µg AIB1 Ab (anti-idiotypic antibody) administration 8 days before tumor induction, compared to that of 1 µg of B(a)P (dose equivalent to that of 500 µg of AIB1 Ab sites).
The preventive effects of a treatment involving a monoclonal anti-idiotypic antibody (Ab) compared to a hormetic model were evaluated on a benzo(a)pyrene [B(a)P]-induced malignant sarcomas in Sprague-Dawley (SD) female rats. This Ab was an anti-idiotypic Ab, internal image of conjugated B(a)P, called AIB1 Ab. It was administered to female rats (500 µg of AIB1 Ab at the top of the left thigh) 8 days prior to tumor induction. Other animals received an injection of 1µg of B(a)P, dose equivalent to that of 500 µg of AIB1 Ab sites. Anti-phosphatidylinositol (PtdIns) autoantibody (autoAb) levels observed in AIB1 Ab-treated rats sera were 70% weaker than those observed in rats treated with 1 µg of B(a)P or control animals. Conversely, no differences were noted in the profil curves between the control group and the group treated with 1µg of B(a)P prior to tumor induction dose. There was a delay of 16 days in tumor onset between 75% of AIB1 Ab-treated rats and the controls and, the mean survival of rats treated with AIB1 Ab was increased compared to the control group. Conversely, there was no delay in tumor onset between rats treated with 1µg of B(a)P and controls. These results revealed that the preventive effects of 1 µg of B(a)P injected prior to tumor induction were lower than those of the monoclonal anti-idiotypic Ab, internal image of B(a)P.
3.1. MONOCLONAL Antibody
The monoclonal AIB1 Ab used in this study was IgG1 (k chain). This monoclonal anti-anti-conjugated B(a)P Ab, has been previously characterized as the internal image of conjugated B(a)P (Chagnaud et al., 1993a). Producing hybridomas were propagated in the peritoneal cavity of Balb/c mice after prior injection with Freund's incomplete adjuvant (Difco, 0.5 ml/mouse). Mouse ascitic fluids were harvested 8-10 days later and monoclonal Ab partially purified by a 50% (NH4)2SO4 precipitation followed by gel filtration (Sephadex G-200).
3.2. SARCOMA MODEL
Eleven female SD rats (Janvier, Le Genest Saint-Isle, France) were housed under controlled conditions (22°C, monitored light-dark cycles, with light from 8:00 a.m. to 8:00 p.m.), and were supplied with food (UAR, Versailles, France) and water ad libitum. After preventive treatment (see below), the 50-60 day-old female SD rats weighing 180-200 g received under anesthesia a single s.c. injection of 2 mg of B(a)P diluted in 500 µl of sesame oil, at the top of the right thigh. Blood of the treated rats was regularly sampled and tested (Figure 1).
3.3. TREATMENT
Ab solution were prepared as follows : G-200 purified fractions of Ab were diluted at 2 mg/ml in NaCl 9‰ (Meram) and stored at - 80°C. Eight days before tumor induction, female SD rats were devided in 3 groups. The first group (n=4) received at the top of the left thigh only one s.c. injection of 500 µg of AIB1 Ab. The second group (n=4) was injected with 1 µg of B(a)P (dose equivalent to that of 500 µg of AIB1 Ab sites). A control group has received an equivalent volume of solvent (n=3). Eight days later, a s.c. injection of 2 mg of B(a)P diluted in sesame oil was administered to all SD female rats at the top of the left thigh (Figure 1).
Figure
1. Description of the experimental protocol : Time sequence of the
events (Pre-treatment, tumor induction, time-course of circulating anti-PtdIns
autoAb).
3.4. Immunoenzymatic assay
Detection of anti-PtdIns autoAb was performed with an adapted ELISA method as previously described by Faiderbe et al. (1992b) on rat sera diluted at 1/1000. Experimental absorbance values obtained from well-plates coated with PtdIns were corrected by subtracting blank values read from wells coated with control protein (thyroglobulin).
3.5. TUMOR GROWTH MEASUREMENT
B(a)P-induced tumors were mechanically measured with a calliper rule as described by Faiderbe et al. (1992b). Their volume was calculated using a standard formula : width2 x length x 0.52, according to Ingber et al. (1990), and expressed as cubic centimeters.
3.6. Histological analysis
Some animals were randomly chosen in each group and their tumors were removed, cut in pieces and fixed in Bouin de Hollande.
4.1. EFFECTS OF PRE-TREATMENT ON ANTI-PHOSPHATidyLINOSITOL AUTOANTIBODY LEVELS
Detection of
anti-PtdIns autoAb was performed as previously described in Material and Methods
in order to monitor the malignant transformation and effects of AIB1 Ab (500
µg) or B(a)P (1 µg) pre-treament (Figure 2).
Figure 2. Effects of AIB1 Ab or B(a)P
pre-treatment on circulating anti-PtdIns autoAb. Optical density (O.D.)
represents the binding of rat autoAb (1/1000) to PtdIns coating on well-plates.
Curves show mean O.D. values with standard error : curve 1 (p), AIB1 Ab pre-treatment
(500 µg) 8 days before tumor induction (2mg of B(a)P) (n=4) ; curve 2 (m),
B(a)P pre-treatment (1 µg) 8 days before tumor induction (n=4); curve 3 (n),
solvent-treatment (control group, n=3).
When AIB1 Ab was administered 8 days before carcinogen injection (2mg of B(a)P, tumor induction), anti-PtdIns autoAb levels were around 70% weaker (curve 1, Figure 2) than those found in rats treated with 1µg of B(a)P (curve 2, Figure 2), or control animals (curve 3, Figure 2). Anti-PtdIns autoAb remained at a low level (70% of control group) until day 140. From that day onwards, autoAb level increased (50% of control group level), and seemed to reach those of control group. No differences were noted in the profil curves between the control group (curve 3) and the group treated with 1µg of B(a)P (curve 2) prior to the tumor-induction dose.
4.2. EFFECTS OF PRE-TREATMENT ON TUMOR GROWTH EVOLUTION
When tumors appeared, they were mechanically measured as previously described in Material and Methods. Figure 3 shows the increase of tumor growth for the three groups treated with AIB1 Ab (500 µg), B(a)P (1 µg), or solvent (control group).
Figure
3. Time-course of tumor growth in rats pre-treated with:
curve 1 (p), 500 µg of AIB1Ab (n = 4) ; curve
2 (n), 1 µg of B(a)P (n = 4) ; curve 3 (m), solvent (n =3). The tumor volume
was calculated with the formula
width2 x length x 0.52.
Ten days after detection of clinically palpable tumors (0.065 cm3), control rat tumor volumes rapidly increased (around day 100, as previously described (Faiderbe et al., 1992b) (Figure 3, curve 3). The animals did not survive beyond day 137.
For SD female rats treated with AIB1 Ab, there was a delay of 16 days in tumor onset between 75% of AIB1 Ab-treated rats and the control group (Figure 3, curves 1 and 3). Moreover, tumor growth volume increased more slowly than those of control group (Figure 3, curves 1 and 3). 75% of animals died between day 140 and day 168. Moreover, 25% of the animals of this group were still alive 168 days after tumor induction, bearing little clinically palpable tumors (0.065 cm3).
For SD female rats treated with 1µg of B(a)P prior to tumor induction, there was no delay in tumor onset between these rats and the animals of the control group (Figure 3, curves 2 and 3). Moreover, tumor growth evolution was slowed down for around 30 days (until day-130). From that day onwards, tumor volumes rapidly increased and became equivalent to that of controls. All the animals treated with 1µg of B(a)P died around day 160.
4.3 EFFECTS OF PRE-TREATMENT ON ANIMAL SURVIVAL
Effects of AIB1 and B(a)P pre-treatment on animal survival have been evaluated.
Figure
4. Effects of AIB1 Ab (500
µg) or B(a)P (1 µg) on animal survival. Cumulative proportion surviving in the
AIB1 Ab-treated rat group, B(a)P-treated rat group and control group was
expressed as percentage of surviving animals during the time-course of experimentation.
(1) AIB1 Ab-treated rats (n =3) ; (2) B(a)P-treated rats (n =4) ; (3) control
group (n =3).
Survival curves in figure 4 showed that the mean survival of the AIB1 Ab-treated rats (curve 1) was increased by about 34 days for 75% of animals (25% are still alive) relative to the control group (curve 3). For rats treated with 1µg of B(a)P, the survival was increased by about 20 days relative to the control group (curve 2).
4.4 HISTOLOGICAL ANALYSIS
Histological analysis of the tumors showed the presence of highly malignant sarcomas with fusiform cells for control rats as previously described by Faiderbe et al., (1992 b) and sarcomas with cells presenting no morphological changes whatever the Ab (500 µg of AIB1 Ab) or carcinogen (1 µg of B(a)P) pre-treatment used.
Using our tumor model induced by a single dose of 2 mg of B(a)P, increased anti-PtdIns autoAb titers were found in all B(a)P-treated SD female rats. All the animals developed a highly malignant sarcoma at the carcinogen injection site (top of the right thigh) around 100 days after administration of 2 mg of B(a)P diluted in sesame oil (Faiderbe et al., 1992b). Tumor incidence and/or growth were easily monitored prior to death. This model was thus reliable for the evaluation of Ab treatment effects. To examine the possible vaccine effects of AIB1 Ab treatment, a single administration of AIB1 Ab before tumor induction was assayed, compared to administration of a single dose of B(a)P (Figure 1). When 500 µg of AIB1 Ab was injected 8 days before tumor induction (injection of 2 mg of B(a)P), anti-PtdIns autoAb levels were 70% weaker in relation of control group (Figure 2, curves 1 and 3). This low level remained stable until day 160 where it slowly increased. Moreover, tumor growth slowed down in relation to that of control group. Animal survival was increased by about 34 days relative to the controls (Figure 4, curve 1). The effects of AIB1 Ab injected prior to tumor induction, confirm the interaction between metabolic pathways involving B(a)P ligand/ cytosolic receptor binding, and the PtdIns as both second messenger and membran component. Comparatively similar effects were observed when AIB1 Ab was administered after tumoral induction (Chagnaud et al., 1993c, 1994).
Anti-PtdIns autoAb levels decreased around 80% in relation to control group when AIB1 Ab was administered biweekly 73 days after carcinogen injection. This decrease was only of 30% when AIB1 Ab was administered biweekly after tumor appearance. For SD female rats treated from day 73 a significant delay of 30 days in tumor onset and a significant mean survival of 35 days were observed. For curative studies on SD female rats bearing little tumors (0.065 cm3) a mean survival of 32 days was observed. Moreover, in 25% of the SD female rats pre-treated with AIB1 Ab tumor growth evolution was not observed even a long time after tumor induction (10 months over).
Whatever the period of AIB1 Ab treatment (preventive or curative) a slowing down in tumoral growth was observed and AIB1 Ab-treated rats survived longer than solvent treated rats. But what is notewhorthy in preventive treatment is that a single injection was necessary to obtain results equivalent to those obtain with repetitive injections in animals treated at differents times after tumor induction.
These results confirmed the capacity of a monoclonal anti-idiotypic Ab, internal image of a ligand involved in tumoral processes, to slow down tumor onset and growth, and to increase animal survival.
Conversely, for SD female rats treated with 1µg of B(a)P, autoAb levels were not modified relative to the control group (Figure 2, curves 2 and 3) and there was no delay in tumor appearance between these rats and the control group (Figure 3, curves 2 and 3). The tumor growth was slowed down for around 30 days before getting equivalent to that of controls (Figure 3, curves 2 and 3). It seemed that the preventive effects of 1 µg of B(a)P injected prior to tumor induction were lower than those of 500 µg of AIB1 Ab.
This preliminary study on preventive effects opens new perspectives. Using larger experimental groups and statistical studies the results will be further convincing.
Acknowledgements : The present work was supported by grants from Dolisos Laboratories (Paris) and IDRPHT (Talence).
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