Int zyxwvutsrqpon J Cancer. zyxwvutsrqpo 50,474-480 (1992) zyxwvutsr Publication of the International Union Against Cancer Publicationde zyxwv I Union Internationale Contre le Cancer zy 0 1992 Wiley-Liss, Inc. INFLUENCE OF DOSE AND SCHEDULE ON THE THERAPEUTIC EFFICACY OF "lI-LABELLED MONOCLONAL ANTIBODY 139H2 IN A HUMAN OVARIAN CANCER XENOGRAFT MODEL Carla F.M. MOLTHOFF', Herbert M. PINEDO, Hennie M.M. SCHLUPER and Epie BOVEN Department of Oncoloa, Free UniversityHospital, De Boelelaan 11 17, 1081 HVAmsterdam, The Netherlands. The therapeutic efficacy of various doses and schedules of '"I-labelled anti-episialin monoclonal antibody (MAb) I39H2 was assessed in the NIH:OVCAR-3 human ovarian cancer xenograft model. Radioimmunotherapywas started at the time S.C. tumors were well established (100 to 300 mm'). The anti-tumor effects induced by i.v. injections of "'I-MAb 139H2 were dose- and schedule-dependent. Optimal growth inhibition and long-lasting complete tumor regressions were obtained with 2 injections of 500, 700 or 750 pCi "'I-MAb 139H2 per mouse given with a 2-week interval. The percentage of tumors with more than 50% reduction of their initial volume after treatment with a total dose of 1,000 pCi '"I-MAb 139H2 per mouse, given as I0 injections of I00 pCi (3 x /week), 4 injections of 250 pCi (2 x/week), 10 injections of 100 pCi (5 x/week) within a period of 3 weeks, or 2 injections of 500 pCi with a 2-week interval, was 9%, 40%. 64% and 75% respectively. Unlabelled MAb I39H2 did not affect tumor growth, while the effects of "'I-control MAb were minor and transient. %MAb 1391.12 treatment did not select for outgrowth of episialin- negative cells in the OVCAR-3 xenografts. Highest absorbed doses of whole-bodyradiation were calculated for 2 injections (500 to 750 pCi "'I-MAb 139H2) given with the 2-week interval. The radiation dose to the tumor after a single injection zyxwvut of 500 pCi "'I-MAb 139H2 was 1,300 cGy over 7 days, which appeared slightly lower than the dose calculated after administrationof a tracer dose of iodinated MAb I39H2. Monoclonal antibodies (MAbs) to tumor-associated anti- gens can be generated relatively easily and in large quantities using hybridoma technology. As a result, these antibodies have been the subject of several investigations in the diagnosis and therapy of human cancers. Immunolocalization of various tumor types has clearly illustrated the capacity of radiolabelled antibodies to visualize tumor lesions in patients (Schlom, 1986). The valuc of radioimmunothcrapy in patients is pres- ently under investigation. For lymphoma as well as melanoma and ovarian cancer, preliminary clinical trials with high doses of '"I-labelled MAbs have already shown promising anti- tumor effects (Press et al., 1989; Divgi and Larson, 1989; Thor and Edgerton, 1989). Howcvcr, the relationship of the dose and schedulc to the outcome of trcatmcnt has yet to be determined. Several MAbs have been incorporated into clinical trials for immunoscintigraphy, eg., HMFG-1, HMFG-2, AUA1, H17E2, OC125 and OV-TL3 (Thor and Edgerton, 1989). It is not clear which of the MAbs will be most useful for radioimmunother- apy. In zyxwvutsrqp a previous study we compared the reactivity of these and additional antibodies in human ovarian cancer xenografts (Molthoff et al., 1991~). We selected MAb 139H2 as one of the most reactive MAbs for further in vivo analysis. MAb 139H2 binds to an epitope of cpisialin, a mucin protein present in most carcinomas (Hilkens et al., 1989). We demonstrated specific uptake and rctention of MAb 139H2 in human tumor xenografts expressing cpisialin (Molthoff et al., 1991b). To date, high doses of radiolabelled MAbs have demon- strated anti-tumor activity in a variety of human tumor xenografts (Wessels, 1990). As the intrinsic radiosensitivity of the tumors is preserved, these models may give additional information on the most optimal radioimmunotherapy sched- ules in cancer patients. Therefore, we designed the present experiments in the human ovarian cancer xenograft model N1H:OVCAR-3 to assess the influence of various doses and schedules on the therapeutic effects of li1I-labelled MAb 139H2. Dosimetry was carried out to obtain insight into the absorbed doses of whole-body-radiation and the possible accumulation of absorbed doses after repeated injections. We also estimated the radiation dose delivered to tumors and organs after injection of a therapeutic dose of 500 pCi "'I-labelled MAb 139H2 and compared the results with those calculated from a tracer dose experiment. MATERIAL AND METHODS Monoclonal antibodies Production and biochemical characterization of MAb 139H2 have been reported earlier (Hilkens, 1988). Ascitic fluid containing MAb 139H2 was kindly provided by Dr. J. Hilkens (Netherlands Cancer Institute, Amsterdam). MAb 139H2 is of the IgG, isotype and binds to a protein determinant of episialin. Purification of the antibody was performed by affinity chromatography using Affi-Gel Protein-A MAPS I1 (Bio-Rad, Utrecht, The Netherlands). MAb A2C6 is also of the IgG, isotype and has been used as a control antibody. MAb A2C6 reacts with the hepatitis-B surface antigen (Zurawski et al., 1983) and was kindly provided by Dr. S.O. Warnaar (Centocor, Leiden, The Netherlands). Radiolabelling MAbs 139H2 and A2C6 were labelled with Ii1I by the iodogen method (Haisma et al., 1986). Free iodine was removed by incubation of the product with an anion-exchange resin suspension in PBS containing 1% BSA (AGl-X8, Bio- Rad). The percentage of binding was determined by TCA precipitation. Specific activities of the iodinated MAbs ranged from 2 to 5 mCi/mg. The immunoreactive fraction of the labelled MAb 139H2 was determined on OVCAR-3 cells according to Lindmo et al. (1984) and was always between 65 and 75%. The OVCAR-3 xenografi model The in vitro cell line OVCAR-3 (N1H:OVCAR-3), kindly provided by Dr. T.C. Hamilton (Fox Chase Cancer Center, Philadelphia, PA) was originally established from the malig- nant ascites of a patient with poorly differentiated adenocarci- noma of the ovary (Hamilton et al., 1983). A transplantable tumor line was established by injecting lo7 cells S.C. in both flanks of female, 8- to 10-week-old NMRI/Cpb nude (nuinu) mice (Harlan, Zeist, The Netherlands). The doubling time of the tumor volume of OVCAR-3 xenografts is 8 to 12 days. Histology shows a poorly-to-moderately differentiated serous adenocarcinoma. Indirect immunoperoxidase staining with MAb 139H2 revealed a heterogeneous reaction pattern with staining detectable mainly in the better differentiated areas of the tumor and at the apical cell membrane (Molthoff et al., 1991a). No staining was detectable with MAb A2C6. 'To whom correspondence and reprint requests should be ad- dressed. Received: July 17,1991 and in revised form September 9, 1991.