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Clinical Cancer Research Vol. 6, 3334-3341, August 2000
© 2000 American Association for Cancer Research

Experimental Therapeutics, Preclinical Pharmacology

Synergistic Interaction between Anti-p185HER-2 Ricin A Chain Immunotoxins and Radionuclide Conjugates for Inhibiting Growth of Ovarian and Breast Cancer Cells That Overexpress HER-21

Fengji Xu, S. Anthony Leadon, Yinhua Yu, Cinda M. Boyer, Kathy O’Briant, Kristine Ward, Amanda McWatters, Xiaoguang Zhao, Duk Soo Bae, Karen DeSombre, Michael R. Zalutsky and Robert C. Bast, Jr.2

University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030 [F. X., Y. Y., K. W., A. M., R. C. B.]; University of North Carolina, Chapel Hill, North Carolina 27514 [S. A. L.]; and Duke University Medical Center, Durham, North Carolina 27710 [C. M. B., K. O., X. Z., D. S. B., K. D., M. R. Z.]


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Radionuclide conjugates or ricin A chain (RTA) immunotoxins that target pl85HER-2 have partially inhibited the growth of human ovarian cancer xenografts in athymic mice but generally have not cured mice bearing human tumor transplants. The present study was undertaken to explore whether a combination of ionizing radiation and an immunotoxin could exert additive or synergistic cytotoxicity in culture and in vivo against cancer cells that overexpress pl85HER-2. In cell culture, treatment with 200–2000 cGy external beam irradiation followed by incubation with TA1-anti-pl85HER-2-RTA immunotoxin (TA1-RTA) produced synergistic inhibition of clonogenic growth of ovarian and breast cancer cells that expressed >106 pl85HER-2 receptors/cell. The effect on cell survival correlated with an inhibition of DNA repair. A prior study (F. J. Xu et al., Nucl. Med. Biol., 24: 451–460, 1997) compared the biodistribution of radionuclide conjugates prepared with monoclonal antibodies that bind to different epitopes on the extracellular domain of pl85HER-2 and found optimal tumor uptake with the 520C9 antibody, which did not compete with TA1 for binding to the receptor. In this report, the TA1-RTA immunotoxin and the 131I-labeled 520C9 radionuclide conjugate could each inhibit the growth of clone-9002-18 xenografts in athymic mice but did not yield long-term survivors using maximally tolerated doses of each agent. When TA1-RTA and 131I-labeled 520C9 were used in combination, a greater inhibition of tumor growth was obtained than with either single agent. Similarly, survival with the combined treatment was significantly prolonged (P = 0.004) relative to treatment with immunotoxin or radionuclide conjugate alone. After treatment with an optimal combination of immunotoxin and radionuclide conjugate, 50% of mice survived >300 days, whereas controls succumbed with a median survival of 36 days. These results suggest that combinations of immunotoxins and radionuclide conjugates deserve further evaluation for the treatment of cancers that overexpress pl85HER-2.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Over the last two decades, monoclonal antibodies and their conjugates have begun to contribute to the management of several forms of human cancer. Treatment with unconjugated antibodies has produced objective regression in a fraction of leukemias (1) , lymphomas (2 , 3) and breast carcinomas (4) . An anti-p185c-erbB-2 (anti-pl85HER-2) antibody has potentiated the activity of cytotoxic drugs in patients with recurrent breast cancer (5) . The therapeutic potency of antibodies has been increased by conjugation with radionuclides. Using radioimmunotherapy, durable responses have been achieved in lymphomas that had proven refractory to conventional agents (6 , 7) . Phase II clinical trials suggest that 90Yttrium- labeled anti-human milk-fat-globule protein (HMFG1) antibody can produce objective responses in patients with epithelial ovarian cancer (8) . At present, the impact on survival of the i.p. administration of 90Yttrium-labeled anti-human-milk-fat-globule protein antibody is being evaluated in a multinational Phase III trial in patients who have had a complete clinical response to conventional therapy.

To improve the clinical efficacy of radioimmunotherapy, radionuclide conjugates might be used in combination with immunotoxins that recognize different antigens or epitopes expressed on the same cancer cells. Immunotoxins have potentiated the cytotoxic activity of alkylating agents (9) and might also potentiate radiation damage. Synergy has also been observed between paclitaxel and radioimmunotherapy with yttirium-90-labeled chimeric antibody (10) . Among the antigenic targets associated with breast and ovarian cancers, pl85HER-2 is overexpressed in up to 30% of cases, and this can be associated with a poor prognosis (11 , 12) . Availability of multiple antibodies that react with antigenically distinct epitopes on the extracellular domain of pl85HER-2 has permitted the targeting of a single receptor by multiple therapeutic approaches including antibody alone, radionuclide conjugates, and immunotoxins.

Unconjugated antibodies against some, but not all, epitopes on the extracellular domain of pl85HER-2 can inhibit clonogenic growth of cells that overexpress the receptor (13) . At optimal concentrations of unconjugated antibody, however, only 90% inhibition of clonogenic growth can be achieved. Treatment with anti-pl85HER-2 antibodies that have been conjugated with RTA can inhibit growth by 99.99%, i.e., a reduction by some 4 logs of clonogenic tumor cells (14) . Optimal cytotoxicity of anti- pl85HER-2-RTA conjugates depends critically on the density of pl85HER-2 receptors on each tumor cell. Tumor cells with >106 copies of pl85HER-2 were most markedly inhibited, whereas tumor cells with 105 copies exhibited less than one log of inhibition (14) . Because normal nonmalignant tissues exhibit, at most, 104 copies of pl85HER-2 per cell, a therapeutic window might exist for treatment with immunotoxins in vivo.

In vitro and in vivo models have been established to test the activity of immunotoxins and radionuclide conjugates that target cells with a high density of pl85HER-2. SKOv3 ovarian cancer cells have been transfected with additional copies of HER-2, and clones have been isolated that express >106 copies of pl85HER-2. Clone-9002-18 cells overexpress pl85HER-2 and have retained the ability to grow both in cell culture and as xenografts in athymic nu/nu mice. Using clone-9002-18 we have asked: (a) whether a combination of external beam radiation and immunotoxin might have additive or synergistic activity against cells that overexpress pl85HER-2; (b) what mechanism(s) might contribute to this interaction; and (c) whether similar additive or synergistic interactions might occur in vivo between anti-pl85HER-2-RTA3 immunotoxin and anti-pl85HER-2 radionuclide conjugates.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Monoclonal Antibodies.
Murine monoclonal antibodies TA1 and 520C9 that react with the extracellular domain of pl85HER-2 were obtained, respectively, from Applied BioTechnology/Oncogene Science (Cambridge, MA; Ref. 15 ) and from Chiron, Inc. (Emeryville, CA; Refs. 16 , 17 ). All of the antibodies were of the IgG1 isotype. MOPC21 (IgGl), an isotype-matched control, did not bind to pl85HER-2. To prepare antibodies, hybridoma cells were washed free from serum and injected i.p. into pristane-primed BALB/c mice. When tense ascites had formed, fluid was harvested aseptically. IgG antibodies were purified from hybridoma-induced ascites fluid using protein A-Sepharose chromatography (Pharmacia LKB, Uppsala, Sweden). Fractions containing protein measured by absorbance at 280 nm were dialyzed for 24 h against 50 mM phosphate buffer (pH 7.4) and were concentrated using an Amicon filter and compressed nitrogen gas. Immununoglobulin concentration was calculated by dividing absorbance at 280 nm by the extinction coefficient for IgG. Immunoglobulin purity was confirmed by SDS PAGE. Purified immunoglobulin was aliquoted and stored at -70°C.

Radioiodination of Monoclonal Antibodies.
Monoclonal antibodies were labeled with Na131I using the iodogen method (18) . In brief, 50 µl of phosphate buffer [0.5 M (pH 7.4)] was added to a 15 x 75-mm borosilicate tube coated with 10–100 µg of iodogen (Pierce Chemical Co. Rockford, IL). Monoclonal antibody (50–500 µg) was added in a volume of 95 µl of PBS [50 mM phosphate buffer and 0. 15 M NaCl (pH 7.4)]. Radioiodination was initiated by the addition of 0. 5–10 mCi of Na131I, and the mixtures were incubated for 30 min on ice. The protein-bound iodine was separated from free 131I by gel filtration on a PD-10 column (Pharmacia, Pleasant Hill, CA) equilibrated with PBS. A sample of 3 µl from each fraction was counted in a Packard gamma counter (Packard Instrument Company, Downers Grove, IL) to measure protein-bound radioactivity. Iodination efficiency ranged between 75 and 90%, and the specific activity was >6 µCi/µg.

Preparation of Immunotoxin.
The TA1 and 520C9 murine monoclonal antibodies were conjugated with RTA using 2-iminothiolane as described previously (19) .

Cell Line.
SKOv3 9002-18 (clone-9002-18) ovarian cancer cells and SKBr3 breast cancer cells were maintained in TCM, consisting of McCoy 5A medium supplemented with 10% FBS, 2 mM L-glutamine, 100 units/ml penicillin, 100 µg/ml streptomycin, and 400 µg/ml G418 (Life Technologies, Inc., Grand Island, NY). Clone-9002-18 cells were produced by transfection of the full-length human HER2/neu (c-erbB-2) gene into the SKOv3 cell line, to provide a subline that expressed 2 x 106 pl85HER-2 binding sites per cell (14) .

Serial Dilution Clonogenic Assay and Isobolographic Analysis.
Cytotoxicity was evaluated using a limiting dilution technique as described previously (20) . After trypsinization, 106 tumor cells were irradiated and/or incubated with immunotoxin for 3 h in a total volume of 1 ml. A Cesium-137 irradiator was used for external beam irradiation delivering 27 cGy/min for total doses <200 cGy and 3904 cGy/min for doses >=200 cGy. Cells were then washed twice with TCM. A series of nine 5-fold dilutions were prepared. Six aliquots (100 µl) of each dilution were plated in 96-well flat-bottomed microtiter plates preloaded with 100 µl of TCM. Plates were incubated for 14 days at 37°C, in 5% CO2 and 95% humidified air. Growth of colonies (>50 cells) was evaluated by visual scoring. Each value was calculated from a mean of duplicate plates. Limiting-dilution analysis was then performed (20) .

Isobolographic analysis, a geometric method to explore drug interactions, was performed as described by Berenbaum (21) and Steel and Peckham (22) . Isoboles for different levels of cytotoxicity were drawn from dose-response curves, in which the log effect by dose of one agent was plotted for each constant dose of the other agents in the combination. The calculation of an "envelope of additivity" between modes I and II, which indicated the theoretical limits of the additive effects obtained from an interaction of two agents. An interaction was considered to be synergistic when the combined cytotoxic effects exerted by two different agents fell below the envelope. In the case of the MOPC-RTA control, traditional isobolographic analysis could not be used to compare the combined effects of radiation and MOPC-RTA because the effect of increasing doses of MOPC-RTA on cell survival was not monotonely decreasing. Consequently we asked whether there was evidence of decreased (or increased) clonogenic growth with increasing concentrations of the immunotoxin at different levels of irradiation. Differences in log surviving fraction between that found at zero concentration of immunotoxin and at levels of 0.1, 0.25, 0.5, and 1.0 µg/ml of MOPC-RTA were fitted by linear regression (23) for each fixed level of radiation. Thus the differences in log cell survival were fitted as:

where SF(R,D) is the surviving fraction at the radiation level R with MOPC-RTA at dose D and where a and b are constants. For each radiation dose, the slope b and the uncertainty in the estimate of b were calculated and compared.

Measurement of Repair Synthesis.
DNA of cultured cells was prelabeled by growing cells for 7 days in medium containing 0.01 µCi/ml [14C]thymidine. For repair analysis, [14C]thymidine-prelabeled cultures were incubated for 1 h before treatment in 10 µM BrdUrd and 1 µM FdUrd, washed with PBS, and irradiated or sham-irradiated with 600 or 2000 cGy of {gamma} irradiation at a dose rate of 1 Gy/min. After irradiation, the cells were allowed to repair in medium containing 10 µM BrdUrd, 1 µM FdUrd, and 30 µCi/ml [3 H]thymidine (82 Ci/mmol) in the presence or absence of the immunotoxin. After 4 h, the medium was removed from the plates, the cultures were washed twice with PBS, and the cells were lysed in 10 mM Tris-HCI (pH 8.0) and 10 mM EDTA with 0.5% SDS.

Repair synthesis was measured as described by Smith et al. (24) by first resolving parental density DNA (containing [3 H]thymidine and BrdUrd substituted repair patches) from hybrid density DNA (synthesized by semiconservative replication) by centrifugation in CsCl gradients. The parental density DNA was further purified in a second neutral CsCl gradient. 14C activity was assayed and the DNA concentration determined spectrophotometrically by measuring the absorbance at 260 nM. 14C specific activity was calculated from the same material, permitting conversion of 14C activity in double-labeled samples to µg DNA before plotting the ratio of 3 H-labeled cpm:µg of DNA. Repair synthesis was calculated from the isolated parental density DNA as the ratio of [3 H]thymidine cpm per µg of DNA.

Growth Inhibition of Clone-9002-18 in nu/nu Mouse Xenografts.
Aliquots of 2 x 107 clone-9002-18 cells that had been grown in tissue culture were trypsinized, washed, and injected s.c. into 20-g athymic BALB/c nu/nu mice (Charles River) in a volume of 0.1 ml. Palpable tumors generally formed 4–5 days after injection. Animals were divided into groups of 5–6 mice. On days 5, 6, and 7 after the injection of tumor cells, TA1-RTA immunotoxin (50 or 75 µg/day) was injected i.p. in a volume of 0.5 ml HBSS into some groups. On day 5 after injection of tumor cells, 131I 520C9 (100 or 250 µCi) was administered i.v. to some groups. The radiation dose received by tumor from 131I-520C9 was estimated from a previous study (25) in which the tissue distribution of this labeled antibody was determined as a function of time. From these data, the tumor-absorbed dose was calculated using standard MIRD formulation. Control groups received injections of HBSS by the same routes and according to the same schedules. Tumor size was measured as the product of two perpendicular diameters every 3 days. Mice were killed when tumor size reached 2 cm or when they appeared to be in distress.

Statistical Analysis.
The significance of differences in tumor size was determined with the Student t test. Differences in survival were evaluated with the Wilcoxon Mann-Whitney rank order test.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Synergistic Interaction between Anti-pl85HER-2 Immunotoxin and Ionizing Radiation in Cell Culture.
In previous studies, the potency of several immunotoxins had been compared after conjugating RTA with murine monoclonal antibodies against different epitopes on the extracellular domain of pl85HER-2. Optimal inhibition of clonogenic growth was observed with TA1-RTA (26) . Cytotoxicity depended on the concentration of immunotoxin (26) and on the density of p185HER-2 expression (14) . When clone-9002-18 ovarian cancer cells that expressed >106 p185HER-2 receptors/cell were tested, an optimal concentration of TA1-RTA immunotoxin (5 µg/ml) could inhibit growth of clonogenic tumor cells by 3.8 logs (Fig. 1Citation A). Treatment with a mixture of unconjugated TA1 (4.2 µg/ml) and free RTA (0.8 µg/ml) inhibited only 0.1 log of clonogenic growth in an experiment in which TA1-RTA (5 µg/ml) reduced clonogenic cells by 3.8 logs (data not shown).



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  		Fig. 1. Inhibition of clonogenic growth of clone-9002-18 cells that overexpress p185HER-2: A, by treatment for 3 h with TA1-RTA (0.1–5.0 µg/ml); B, by {gamma} irradiation (20–2000 cGy); or C, by {gamma} irradiation followed by incubation for 3 h with TA1-RTA immunotoxin. Data reflect the mean log reduction in clonogenic growth ± SE for two to six replicate experiments.

 
To evaluate interactions of ionizing radiation and immunotoxin, we have treated clone-9002-18 cells ovarian cancer cells with different doses of {gamma} radiation before incubation for 3 h with different concentrations of TA1-RTA. As would be anticipated with mammalian cells, inhibition of clonogenic growth was observed in a log-linear dose-dependent manner after treatment with 20–2000 cGy (Fig. 1Citation B). When clone-9002-18 cells were treated with a combination of immunotoxin and {gamma} radiation, increasing concentrations of TA1-RTA immunotoxin (0.1–1.0 µg/ml) produced progressively greater cytotoxicity in combination with {gamma} irradiation (Fig. 1Citation C). Isobolographic analysis indicated that the two modalities interacted synergistically with an isobole that fell below the envelope of additivity (Fig. 2)Citation . Similar synergy was observed between {gamma} radiation and the 520C9-RTA immunotoxin that recognized a different epitope on the extracellular domain of p185HER-2 (data not shown). Unconjugated TA1 antibody (Fig. 1Citation C), 520C9 antibody (data not shown) or nonspecific MOPC antibody (data not shown) failed to potentiate the response to {gamma} irradiation. Similarly, MOPC-RTA did not interact with radiation. In two replicate experiments (data not shown), MOPC-RTA killed less than 0.5 logs of clonogenic cancer cells over a wide range of concentrations (0.1–1.0 µg/ml). Linear regression analysis was performed for different MOPC-RTA concentrations (0.1–1.0 µg/ml) at each of several levels of radiation (0–2000 cGy). In every case, the slope b was less than the uncertainty of the slope, indicating that there was not enough information to show that there was any interaction between MOPC-RTA and radiation. Interestingly, a 3-h treatment with the protein synthesis inhibitor cyclohexamide (10-2-10-7 M) failed to potentiate the effect of radiation on clonogenic growth (data not shown).



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  		Fig. 2. Isobolographic analysis of the interaction between different doses of {gamma} irradiation and subsequent incubation for 3 h with different concentrations of TA1-RTA immunotoxin before limiting-dilution assay of clonogenic growth. The isobole is plotted for 1.29 log kill. In three of four replicate experiments synergistic interactions were found, and in the fourth, additive interactions were documented.

 
To determine whether synergy would be observed between ionizing radiation and immunotoxin against other cell lines that overexpressed p185HER-2, experiments were performed with the SKBr3 breast cancer cell line that expressed >106 copies of the receptor per cell. Synergistic interactions were observed on isobolographic analysis of experiments with SKBr3 but with less regularity than had been found with clone 18. In all of the experiments with both cell lines, the interaction of radiation and immunotoxin was either synergistic or additive. Synergistic interactions were documented in three of four experiments with clone 18 ovarian cancer cells and in three of seven experiments with SKBr3 breast cancer cells (data not shown). Difficulty in demonstrating a consistently synergistic interaction between radiation and immunotoxin with SKBr3 cells seemed related to the fact that the SKBr3 cell line was more sensitive to radiation than was clone-9002-18. After treatment with 600 cGy in synergy experiments, 0.84 logs of clone-9002-18 were eliminated compared with 2.19 logs of SKBr3 (P = 0.003; Wilcoxon Mann-Whitney rank order test). When lower doses (20–1000 cGy) of radiation were evaluated, synergy was observed in two of three experiments with SKBr3 cells.

Immunotoxin-mediated Inhibition of Radiation-induced DNA Repair.
DNA repair was measured by the incorporation of [3 H]thymidine after irradiation (600–2000 cGy). Repair replication was induced by irradiation in a dose-dependent manner (Fig. 3)Citation . Subsequent incubation with TA1-RTA (0.1–0.5 µg/ml) for 4 h inhibited radiation-induced repair. Higher doses of TA1-RTA produced greater inhibition of radiation-induced DNA repair. Similar results were obtained with clone-9002-18 ovarian cancer cells and with SKBr3 breast cancer cells that also expressed >106 p185HER-2 receptors per cell.



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  		Fig. 3. TA1-RTA immunotoxin-mediated inhibition of DNA repair after ionizing radiation. SKBr3 breast cancer cells (A) or clone-9002-18 ovarian cancer cells (B) were preincubated with [14C]thymidine, BrdUrd, and FdUrd as described in "Materials and Methods." After irradiation (600–2000 cGy), cells were allowed to repair 4 h in medium containing [3H]thymidine, BrdUrd, and FdUrd with or without TA1-RTA immunotoxin (0.1–0.5 µg). After lysis and DNA separation on CsCl gradients, repair synthesis was calculated from the isolated parental density DNA as the ratio of [3H]thymidine cpm per µg of DNA. Results are the average of two independent experiments on different days.

 
Synergistic Antitumor Activity in Vivo Using a Combination of p185HER-2 Immunotoxin and Radionuclide Conjugate.
When RTA immunotoxins were prepared with different anti-p185HER 2 antibodies, optimal inhibition of clone-9002-18 growth in cell culture had been obtained in previous studies with TA1-RTA immunotoxin (26) . To evaluate the impact of TA1-RTA treatment in vivo, clone-9002-18 ovarian cancer cells were grown as xenografts in athymic BALB/c nu/nu mice. Transplants of 2 x 107 clone-9002-18 cells grew regularly after s.c. injection and formed palpable tumors within 5 days. In a dose-finding study, groups of five mice with s.c. tumor transplants were treated i.p. with TA1-RTA at 50 or 75 µg per day on days 5, 6, and 7 after transplant (data not shown). At the higher dose, one of five mice died of acute toxicity 11 days after transplant and 4 days after the last injection of immunotoxin. In subsequent studies, 50 µg/day on each of 3 consecutive days was considered a maximally tolerated dose of TA1-RTA immunotoxin. TA1-RTA significantly inhibited s.c. tumor growth (P < 0.05) in both groups between days 11 and 28, but complete regression of xenografts was not attained (Fig. 4)Citation . Similar antitumor activity was observed with the two dose levels of TA1-RTA.



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  		Fig. 4. Effect of TA1-RTA immunotoxin on growth of clone-9002-18 xenografts in nu/nu mice. Groups of 6 mice were treated with diluent, TA1-RTA (50 µg/day on days 5, 6, and 7) or TA1-RTA (75 µg/day on days 5, 6, and 7). Compared with the diluent control, tumor growth was inhibited by TA1-RTA (50 µg/day for 3 days) on days 8–28 (P < 0.034 to P < 0.002). Tumor-growth inhibition by TA1-RTA (75 µg/day for 3 days) was observed on days 8–31 (P < 0.024 to P < 0.002). All of the mice exhibited progressive tumor growth. Each bar, the mean tumor diameter ± SE.

 
Because synergistic interactions had been observed between radiation and immunotoxin in cell culture, we evaluated the antitumor activity of TA1-RTA immunotoxin, alone and in combination with a different anti-p185HER-2 antibody that had been labeled with 131I. In earlier studies, radiolabeled 520C9 exhibited optimal tumor targeting of clone-9002-18 xenografts in athymic nu/nu mice (25) . Because TA1 and 520C9 recognized distinct epitopes on the extracellular domain of p185 HER-2 (13) , the two conjugates could be used in combination to evaluate the possibility that additive or synergistic interactions between radiation and immunotoxin might be observed in vivo as well as in cell culture.

Groups of six mice with established tumor transplants were treated with: (a) diluent; (b) 50 µg/day TA1-RTA (days 5, 6, and 7); (c) 131I-labeled 520C9 (100 or 250 µCi on day 5); or (d) both immunotoxin and radionuclide conjugate. For injected activities of 100 and 250 µCi, it is estimated that these xenografts received 766 and 1916 rads, respectively. The 131I-labeled 520C9 inhibited tumor growth at a maximally tolerated dose of 250 µCi/mouse (Table 1)Citation but failed to produce long-term survival in two replicate experiments (Table 2)Citation . When TA1-RTA (50 µg/mouse on days 5, 6, and 7) was combined with 131I-labeled 520C9 (100 or 250 µCi/mouse on day five), significant growth inhibition was attained (P < 0.001). The higher delivered dose of radionuclide (1916 rads) seemed more effective than the lower dose (766 rads) when used as a single agent or in combination with TA1-RTA (Fig. 5)Citation . In the first experiment, all of the mice that were tumor-free at 34 days were followed long-term, and in the second experiment all of the animals were followed for progressive tumor growth. Survival was modestly but significantly prolonged with each of the individual agents (Fig. 6)Citation . When immunotoxin and radionuclide conjugate (100 µCi or 250 µCi) were combined, survival was significantly better (P = 0.004) than that attained with either single agent. The optimal combination of immunotoxin (50 µg TA1-RTA every day for 3 days) and radionuclide conjugate (250 µCi 131I-labeled 520C9) extended median survival from 36 days to 356 days. Biological variation was noted between the two studies, but when both experiments are considered together, survival of >180 days was observed in 8 of 12 mice and survival of >300 days was observed in 6 of 12 (Table 2)Citation .


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  		Table 1 Growth inhibition of s.c. clone-9002-18 ovarian cancer xenografts after treatment with TA1-RTA immunotoxin and 131I-labeled 520C9 radionuclide conjugates, alone and in combinationa

 

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  		Table 2 Long-term survival of nu/nu mice with clone-9002-18 ovarian cancer xenografts after treatment with TA1-RTA immunotoxin and 131I-labeled 520C9 radionuclide conjugate, alone and in combination

Mice were treated as described in Table 1Citation . In experiment 1, mice with measurable tumors were killed on day 34, but mice that had achieved complete regression of tumor transplants were observed up to 300 days. In experiment 2, all of the mice were observed up to 300 days.

 


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  		Fig. 5. Effect of TA1-RTA and 131I-labeled 520C9, individually and in combination, on the growth of clone-9002-18 xenografts in nu/nu mice. Groups of six mice were treated with diluent, TA1-RTA (50 µg/mouse on days 5, 6, and 7), 131I-labeled 520C9 (100 µCi/mouse on day 5), 131I-labeled 520C9 (250 µCi/mouse on day 5), TA1-RTA (50 µg/mouse on days 5, 6, and 7) plus 131I-labeled 520C9 (100 µCi/mouse on day 5), or TA1-RTA (50 µg/mouse on days 5, 6, and 7) plus 131I-labeled 520C9 (100 µCi or 250 µCi/mouse on day 5). Relative to diluent controls, tumor growth was significantly inhibited (P < 0.01) by TA1-RTA, 131I-labeled 520C9 (250 µCi), and the combination of TA1-RTA and 131I-labeled 520C9 (100 µCi or 250 µCi) on days 10, 22, and 31. Significantly greater inhibition (P < 0.05 to P < 0.00002) was observed with a combination of TA1-RTA and 131I-labeled 520C9 (100 µCi or 250 µCi) than with either single agent on days 10, 22, and 31.

 


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  		Fig. 6. Survival after treatment of clone-9002-18 xenografts with saline, TA1-RTA (50 µg/mouse on day 5, 6, and 7), 131I-labeled 520C9 (100 µCi/mouse on day 5), 131I-labeled 520C9 (250 µCi/mouse on day 5), TA1-RTA (50 µg/mouse on day 5, 6, and 7) plus 131I-labeled 520C9 (100 µCi/mouse on day 5), TA1-RTA (50 µg/mouse on day 5, 6, and 7) plus 131I-labeled 520C9 (250 µCi/mouse on day 5). Each group contained 6 mice. Survival was significantly prolonged relative to the diluent in all of the treatment groups (P < 0.01). The combination of TA1-RTA and 131I-labeled 520C9 (100 µCi or 250 µCi) prolonged survival to a greater degree than did either single agent (P = 0.004).

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
This study documents a synergistic interaction between radionuclide conjugates and immunotoxins that target p185HER-2 both in cell culture and in vivo. Despite an extensive preclinical and clinical literature regarding conjugation of monoclonal antibodies with drugs, toxins, and radionuclides (reviewed in Ref. 27 ), we could find no reports that have considered the interaction of immunotoxins with radionuclide conjugates. Mechanisms underlying the synergistic interaction between radiation and immunotoxins are not fully defined. Observations presented above suggest that immunotoxins may interfere with radiation-induced DNA repair. Because RTA inhibits protein synthesis, the inhibition of repair could relate to the depletion of repair enzymes or peptide cofactors that have relatively short half-lives or that are in limiting concentration. Several studies have demonstrated that immunotoxins can induce apoptosis (28 , 29) , and this is true of anti-p185HER-2-RTA.4 The inhibition of protein synthesis alone generally does not induce apoptosis (30 , 31) , but the persistence of unrepaired DNA damage may contribute to the enhancement of programmed cell death after irradiation. In the present study, a 3-h treatment of clone-9002-18 cells with cyclohexinide failed to potentiate the cytotoxicity of ionizing radiation. An RTA immunotoxin may, however, persist within cells for much longer intervals than cycloheximide and may irreversibly inhibit protein synthesis by depurination of ribosomal proteins.

Synergy may relate, in part, to the activation of the HER-2 kinase or to greater internalization of p185HER-2 by the binding of antibodies to two different epitopes. Neither 520C9 nor the TA1 antibody triggers a significant (2-fold) increase in phosphorylation of HER-2 (19) . Moreover, our previous studies have demonstrated that receptor kinase activity is not required for internalization of antibody (32) or for immunotoxin-induced cytotoxicity (19) . Approximately 30% of cell-bound TA1 or ID5 anti-HER-2 antibody can be internalized within 1 h regardless of the ability of the antibody to induce phosphorylation of receptors (13) . Internalization could lead to more rapid dehalogenation that could actually decrease the therapeutic activity of the radionuclide conjugate. The antibody alone fails to produce synergy, which suggests that antibody-induced signaling is not likely to change radiosensitivity. Synergy is also observed with immunotoxin and external beam radiation, which suggests that the binding of antibodies to two different epitopes is not required.

Whatever the mechanism of the observed synergy, the use of immunotoxins and radionuclide conjugates may exert greater antitumor activity in vivo. A synergistic inhibition of growth was observed in cell cultures at relatively high rates of external beam {gamma} radiation (27–3904 rads/min) and in vivo with relatively low rates of delivered dose of high energy ß particles (766–1916 rads/several days). Because of the multicellular range of 131I ß particles, the bystander activity of radiation may help to eliminate cells with relatively poor expression of p185HER-2. Despite the probable importance of p185HER-2 for the induction and maintenance of malignant transformation, substantial heterogeneity has been observed in the expression of the receptor in different areas of the same tumor (33) . The direct or bystander effects of radionuclide conjugates may also eliminate cancer cells that are inherently resistant to immunotoxin. Observations with heterografts suggest that more complete elimination of tumor cells can be attained with the two modalities, permitting long-term disease-free survival in settings in which either single modality is inadequate. Greater efficacy with a single course of treatment may be critical for the use of conjugates that contain highly immunogenic plant or bacterial toxins.

Because BALB/c mice do not express human p185HER-2, the present study does not permit assessment of toxicity that could potentially arise by the targeting of immunotoxins and radionuclide conjugates to a receptor that is expressed on some normal human tissues. Immunohistochemical studies have detected low levels of p185HER-2 in human skin and gastrointestinal mucosa with trace expression in a number of other organs (33) . To the extent that toxicity related to specific binding is dose-related, synergistic interactions between immunotoxins and radionuclide conjugates may permit effective treatment with lower doses of each agent. Studies in cell culture suggest that anti-p185HER-2 will exert optimal toxicity against cells with >106 copies of p185HER-2 per cell and little, if any, toxicity against cells with 104 receptors (14) . Ultimately, toxicity can be assessed preclinically only in primates that express p185HER-2 that can bind RTA-anti-p185HER-2 conjugates.

Ovarian cancer is a particularly attractive target for clinical trials of serotherapy with monoclonal antibodies and their conjugates. Ovarian cancer afflicts 25,000 women in the United States each year and causes some 14,500 deaths annually. Despite advances in surgery and chemotherapy, the cure rate has changed little during the last decade. In the short run, a majority of patients will respond to cytoreductive surgery followed by chemotherapy that includes a platinum compound and a taxane. Approximately 40% of patients with advanced ovarian cancer will have a complete clinical response documented at second-look operations. At least one-half of these individuals will, however, experience recurrence of ovarian cancer, generally within 3 years, and will subsequently die from their disease. In this setting, treatment with radionuclide conjugates and immunotoxins could be justified if evidence of activity in Phase II trials could be obtained. Results of randomized Phase III trials could be evaluated with a relatively short lead time.

In the present study, the impact of immunotoxins and radionuclide conjugates was assessed using s.c. tumor transplants where access of these agents to tumor cells was achieved through the intravascular space. Study of s.c. nodules permitted frequent and precise measurement of the impact of the different treatments on tumor size. Because the progressive growth of ovarian cancer requires neovascularization, the use of this s.c. model may reflect the outcome of disease at several different sites. The distinctive pattern of spread for epithelial ovarian cancer over the surface of the peritoneum predisposes, however, to recurrence within the abdominal cavity in a majority of cases. After an apparently complete clinical response to conventional therapy, microscopic deposits of tumor can remain on the peritoneal surface. Consequently, in future preclinical studies, radionuclide conjugates and immunotoxins will be evaluated also against i.p. tumor transplants.


   ACKNOWLEDGMENTS
 
We greatly appreciate the editorial assistance of Adrienne Mattea.


   FOOTNOTES
 
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 in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by NIH Research Grant CA 39930 from the Department of Health and Human Services. Back

2 To whom requests for reprints should be addressed, at Box 355, University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77030. Back

3 The abbreviations used are: RTA, ricin A chain; TCM, tissue culture medium; BrdUrd, 5-bromodeoxyuridine; FdUrd, fluorodeoxyuridine. Back

4 K. Bozorgyi, L. Pusztai, J. C. Dalrymples, B. McWalters, F. Xu, G. B. Mills, and R. C. Bast, unpublished data. Back

Received 9/ 7/99; revised 5/10/00; accepted 5/17/00.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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