Objectives: Endometrial cancer occurs in ~2.8% of American women, with endometrial adenocarcinoma as the most prevalent form of the disease. While endometrial cancer is treated reasonably effectively with hysterectomy at early stages — albeit via a surgery with lifelong healthy implications — prognoses worsen dramatically for patients with disseminated disease. As a result, new tools for the nuclear imaging and targeted radiopharmaceutical therapy of endometrial cancer could have significant impact on patient care. Herein, we evaluate a trio of antigens — HER2, MUC-16, and CD24 — as targets for the immunoPET and radioimmunotherapy of endometrial adenocarcinoma.

Methods: Three monoclonal antibodies — trastuzumab, AR9.6, and ATG-031 — that target HER2, MUC-16, and CD24, respectively, were obtained from commercial sources. Human endometrial adenocarcinoma cells (HEC-1-A) were cultured under standard conditions and were subsequently subjected to immunocytochemical (ICC) staining and confocal imaging for each of the three antigens. Histological immunofluorescence staining for all three antigens was also performed on patient-derived endometrial adenocarcinoma tissue (n = 5). Next, trastuzumab, AR9.6, and ATG-031 were modified with desferrioxamine (DFO) using p-SCN-Bn-DFO, and the binding of each immunoconjugate to its target antigen was compared to its native analogue via ELISA. The DFO-bearing immunoconjugates were then labeled with [⁸⁹Zr]Zr⁴⁺ under neutral conditions (pH 7.4) and purified via gel filtration, and the serum stability of the resultant ⁸⁹Zr-labeled radioimmunoconjugates was assessed via iTLC and SE-HPLC. Immunoreactivity for all three radioimmunoconjugates was evaluated via a cell-binding assay using HEC-1-A cells. Finally, athymic nude mice bearing subcutaneous HEC-1-A xenografts were administered either [⁸⁹Zr]Zr-DFO-trastuzumab (100 μCi, 5 μg, n = 4), [⁸⁹Zr]Zr-DFO-AR9.6 (100 μCi, 5 μg, n = 4), or [⁸⁹Zr]Zr-DFO-ATG-031 (100 μCi, 5 μg, n = 3) via tail vein injection, and PET images were collected 24, 72, and 120 h thereafter. 

Results: ICC revealed the expression of HER2, MUC-16, and CD24 by HEC-1-A cells, with the latter the most abundant. Patient-derived endometrial cancer tissue also displayed expression of all three antigens. The three immunoconjugates — DFO-trastuzumab, DFO-AR9.6, and DFO-ATG-031 — showed similar binding to their respective targets compared to their native counterparts, underscoring that bioconjugation did not perturb the binding affinity of the parent mAbs. [⁸⁹Zr]Zr-DFO-trastuzumab, [⁸⁹Zr]Zr-DFO-AR9.6, and [⁸⁹Zr]Zr-DFO-ATG-031 were all produced in >95% radiochemical yield and >99% purity with specific activities of ~3 mCi/mg post-purification. [⁸⁹Zr]Zr-DFO-trastuzumab, [⁸⁹Zr]Zr-DFO-AR9.6, and [⁸⁹Zr]Zr-DFO-ATG-031 all proved to be >90% stable in human serum over 5 days. PET images and biodistribution studies revealed that all three radioimmunoconjugates effectively target endometrial adenocarcinoma, with [⁸⁹Zr]Zr-DFO-ATG-031 yielding the highest tumor-to-background activity concentration ratios. A PDX model of endometrial carcinoma is currently in development, and PET studies of this advanced disease model are planned in the near future. 

Conclusions: We have synthesized and characterized three radioimmunoconjugates — [⁸⁹Zr]Zr-DFO-trastuzumab, [⁸⁹Zr]Zr-DFO-AR9.6, and [⁸⁹Zr]Zr-DFO-ATG-031 — that target endometrial adenocarcinoma. In vivo results using a subcutaneous model of the disease suggest that CD24 ([⁸⁹Zr]Zr-DFO-ATG-031) may be the most promising radiotheranostic target.

Acknowledgments: The authors thank the MSKCC Small Animal Imaging Core, Radiochemistry and Molecular Imaging Core, and Antitumor Assessment Core.

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Figure 1. (A) Confocal images of a patient-derived endometrial cancer sample (patient: 0029a) stained for HER2, MUC-16, CD24 (green) and DAPI (blue); (B) Representative coronal (left) and maximum intensity projection (right) PET images obtained following the administration of [⁸⁹Zr]Zr-DFO-trastuzumab, [⁸⁹Zr]Zr-DFO-AR9.6, or [⁸⁹Zr]Zr-DFO-ATG-031 to athymic nude mice bearing subcutaneous HEC-1-A xenografts; (C) Ex vivo biodistribution data collected 120 h after the administration of [⁸⁹Zr]Zr-DFO-trastuzumab, [⁸⁹Zr]Zr-DFO-AR9.6, or [⁸⁹Zr]Zr-DFO-ATG-031. 
 

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