Survey
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
[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 4293 Downloaded from cancerres.aacrjournals.org on August 3, 2017. © 1987 American Association for Cancer Research. 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 4294 l UM* Downloaded from cancerres.aacrjournals.org on August 3, 2017. © 1987 American Association for Cancer Research. 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. 1. Tsukada, Y., Mikuni, M., Watabe, H., Nishi, S., and Hirai, H. Effect of antia-fetoprotein serum on some cultured tumor cells. Int. J. Cancer, 13: 187195, 1974. 2. Wepsic, H. T., Tsukada, Y., Takeichi, N., Nishi, S., and Hirai, H. Effect of horse antibody to rat a-fetoprotein upon the growth of AH66 in Donryu rats. Int. J. Cancer, 25:655-661, 1980. 3. Tsukada, Y., Bischof, W. K-D., Hibi, N., Hirai, H., Hurwitz, E., and Sela, M. Effect of a conjugate of daunomycin and antibodies to rat a-fetoprolein on the growth of a-fetoprotein-producing tumor cells. Proc. Nati. Acad. Sci. USA, 79:621-625, 1982. 4. Tsukada, Y., Hurwitz, E., Kashi, R., Sela, M., Hará, A., and Hirai, H. Chemotherapy by intravenous administration of conjugates of monoclonal and conventional anti-rat a-fetoprotein antibodies. Proc. Nati. Acad. Sci. USA, 79:7896-7899,1982. 5. Odashima, S. Establishment of ascites hepatomas in the rat, 1951-1962. In: T. Yoshida (ed.), Ascites Tumors—Yoshida Sarcoma and Ascites Hepatoma(s). NCI Monograph 16, pp. 51-90, 1964. 6. Tsukada, Y., and Hirai, H. Studies on a-fetoprotein in culture of rat hepatoma cells. In: S. Sell and W. H. Fishman (eds.), Onco-developmental Gene Expression, pp. 639-646. New York: Academic Press, 1976. 7. Hurwitz, E., Levy, R., Marón, R., Wilchek, M., Amon, R., and Sela, M. The covalent binding of daunomycin and Adriamycin to antibodies with retention of both drug and antibody activities. Cancer Res., 35:1175-1181, 1975. 8. Hurwitz, E., Marón, R., Berstein, A., Wilchek, M., Sela, M., and Arnon, R. The effect in vivo of chemotherapeutic drug-antibody conjugates in two murine experimental tumour systems. Int. J. Cancer, 21: 747-755, 1978. 9. Hurwitz, E., Schechter, II.. Amon, R., and Sela, M. Binding of anti-tumor immunoglobulins and their daunomycin conjugates to the tumor and its metastasis. In vitro and in vivo studies with Lewis lung carcinoma. Int. J. Cancer, 24:461-470, 1979. 10. Tsukada, Y., Hibi, N., Hará, A., Hurwitz, E., and Sela, M. Effect of a conjugate of daunomycin and purified polyclonal and monoclonal antibodies to rat a-fetoprotein on the growth of a-fetoprotein-producing tumor cells. Ann. NY Acad. Sci., 417: 262-269, 1983. 11. Tsukada, Y., Mikuni, M., and Hirai, H. In vitro cloning of a rat ascites hepatoma cell lines, AH66 with special reference to a-fetoprotein synthesis. Int. J. Cancer, 13: 196-202, 1974. 12. Mizejewski, G. J., and Allen, R. P. Immunotherapeutic suppression in transplantable solid tumors. Nature (Lond.), 250: 50-52, 1974. 13. Mizejewski, G. J., Young, S. R., and Allen, R. P. a-Fetoprotein: effect of heterologous antiserum on hepatoma cells in vitro. J. Nati. Cancer Insu 54: 1361-1367, 1975. 14. Tsukada, Y., Kato, Y., Umemoto, N., Takeda, Y., Hará,T., and Hirai, H. An anti a-fetoprotein antibody-daunomycin conjugate with a novel poly i glutamic acid derivative as intermediate drug carrier. J. Nati. Cancer Inst., 73:721-729, 1984. 15. Kato, Y., Tsukada, Y., Hara, T., and Hirai, H. Enhanced antitumor activity of mitomycin C conjugated with anti-a-fetoprotein antibody by a novel method of conjugation. J. Appi. Biochem., 5:313-319, 1983. 16. Kato, Y., Umemoto, N., Kayama, Y., Fukushima, H., Takeda, Y., Hara, T., and Tsukada, Y. Improved cytotoxicity of daunomycin when conjugated with anti a-fetoprotein antibody. J. Med. Chem., 27:1602-1607, 1984. 17. Amon, R., and Sela, M. Targeted chemotherapy: drugs conjugated to antitumour antibodies. Cancer Surv., /: 429-449, 1982. 18. Belles-Isles, M., and Page, M. In vitro activity of daunomycin-anti a-fetopro tein conjugate on mouse hepatoma cells. Brit. J. Cancer, 41:841-842, 1980. 19. Ohkawa, K., Tsukada, Y., Hibi, N., Umemoto, N., and Hará,T. Selective in vitro and in vivo inhibition against human yolk sac tumor cell lines by purified antibody against human a-fetoprotein conjugated with mitomycin C via human serum albumin. Cancer Immunol. Immunother.,.'.). 81-86, 1986. 20. Tsukada, Y., Ohkawa, K., and Hibi, N. Suppression of human a-fetoproteinproducing hepatocellular carcinoma growth in nude mice by an anti afetoprotein antibody-daunomycin conjugate with a poly L-glutamic acid derivative as intermediate drug carrier. Brit. J. Cancer, 52:111-116, 1985. 21. Ohkawa, K., Tsukada, Y., Hibi, N., Umemoto, N., and Hara, T. Evaluation of a conjugate of purified antibodies against human AFP-dextran-daunorubicin to human AFP-producing yolk sac tumor cell lines. Cancer Immunol. Immunother., 22:81-86, 1986. 4295 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. Updated version E-mail alerts Reprints and Subscriptions Permissions Access the most recent version of this article at: http://cancerres.aacrjournals.org/content/47/16/4293 Sign up to receive free email-alerts related to this article or journal. To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department at [email protected]. To request permission to re-use all or part of this article, contact the AACR Publications Department at [email protected]. Downloaded from cancerres.aacrjournals.org on August 3, 2017. © 1987 American Association for Cancer Research.