Abstract
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Objectives Novel effective strategies to manage recurrent, chemo-resistant ovarian cancer are urgently needed due to the poor patient prognosis associated with this disease. Strategies that target differentiated ovarian cancer cells and ovarian cancer initiating cells (CICs) are expected to display an increased therapeutic efficacy, since CICs are thought to play a major role in metastatic spread and in disease recurrence. The latter two are the major causes of ovarian cancer patients’ morbidity and mortality. The B7-H3 epitope recognized by monoclonal antibody (mAb) 376.96 was selected as a target due to its high expression on differentiated ovarian cancer cells and on ovarian CICs and its restricted distribution in normal tissues. B7-H3-specific mAb 376.96 was radiolabeled with 212Pb, which serves as an in vivo generator of highly cytotoxic 212Bi alpha-particles. The goal of this study was to investigate the usefulness of 212Pb-mAb 376.96 as a targeted radioimmunotherapeutic (RIT) agent in models of ovarian cancer in vitro and in vivo.
Methods mAb 376.96 was reacted with the chelate TCMC and radiolabeled with 212Pb obtained from an 8 mCi 224Ra/212Pb generator (Oak Ridge National Laboratory). In vitro Scatchard assays gauged the binding characteristics of 212Pb-mAb 376.96 to ES-2 ovarian cancer cells. In vitro clonogenic survival assays were performed with B7-H3-specific 212Pb-mAb 376.96 and irrelevant isotype-matched 212Pb-mAb F3-C25 to determine the effects of targeted/non-targeted 212Pb on ES-2 adherent cells and non-adherent tumorspheres containing enriched CICs. In vivo planar gamma camera imaging and biodistribution following i.p. injection of 212Pb-mAb 376.96 and 125I-mAb F3-C25 were studied in CB17 SCID mice bearing i.p. implanted ES-2 cells. Targeted/non-targeted RIT effects on survival of athymic nude mice bearing i.p. implanted ES-2 cells were assessed following i.p. injection of 212Pb-mAb 376.96 (4.5-13.8 µCi) or 212Pb-mAb F3-C25 (9.0 µCi).
Results 212Pb-mAb 376.96 was produced in high radiolabeling yields and purity (both >95%) as indicated by ITLC analysis. Scatchard plot analysis showed a Kd of 10.6 nM for 212Pb-376.96 and 118,000 B7-H3 sites/cell on ES-2 cells. ES-2 in vitro clonogenic survival was significantly inhibited with 212Pb-mAb 376.96 (IC50 = 0.88 ± 0.08 and 0.81 ± 0.35 µCi/mL for adherent and non-adherent tumorsphere cells, respectively) compared to 212Pb-mAb F3-C25 (3.03 ± 0.10 and 1.92 ± 0.56 µCi/mL for adherent and non-adherent cells, respectively) (p<0.01). The uptake of 212Pb-mAb 376.96 at 24 h in solid tumor tissue (7.4 ± 1.8% ID/g) was greater than that of 125I-mAb F3-C25 (5.3 ± 1.1% ID/g) (p = 0.057). In contrast, both mAbs showed comparable retention in the peritoneal ascitic fluid at 24 h. 212Pb-mAb 376.96 and 212Pb-mAb F3-C25 significantly increased mouse median survival relative to non-treated controls (32-42.5, 28, and 15 days, respectively); survival was significantly greater for 9.5 µCi 212Pb-mAb 376.96 compared to a similar dose of 212Pb-mAb F3-C25 (32 and 28 days, respectively; p<0.05).
Conclusions 212Pb-mAb 376.96 specifically binds to B7-H3 expressing ovarian cancer cells. This RIT agent eliminates both differentiated ovarian cancer cells and ovarian CICs in vitro. Lastly, 212Pb-mAb 376.96 targets ovarian cancer tissue in vivo and significantly prolongs the survival of mice bearing an aggressive model of ovarian cancer. These results suggest that B7-H3-specific 212Pb-mAb 376.96 is a useful reagent to optimize and implement RIT of ovarian cancer. This research was supported in part by NIH R21CA173120.