Assessing the use of tumor-specific DARPin-toxin fusion proteins for ex vivo purging of cancer metastases from human ovarian cortex before autotransplantation

Abstract

Objective: To assess the use of tumor-specific designed ankyrin repeat proteins (DARPins) fused to a domain of Pseudomonas aeruginosa exotoxin A for purging of cancer metastases from the ovarian cortex.

Design: Experimental study.

Setting: University medical center.

Patient(s): Human ovarian cortex.

Intervention(s): Ovarian cortex harboring artificially induced breast cancer metastases was treated with DARPins targeted to epithelial cell adhesion molecule (EpCAM) and human epidermal growth factor receptor 2 (HER2).

Main outcome measure(s): The presence of any remaining cancer cells after purging was analyzed by (immuno)histochemistry and reverse transcriptase polymerase chain reaction. Effects on the viability of the ovarian cortex were determined by (immuno)histology, a follicular viability assay, and an assay to determine the in vitro growth capacity of small follicles.

Result(s): After purging with EpCAM-targeted DARPin, all EpCAM-positive breast cancer cells were eradicated from the ovarian cortex. Although treatment had no effect on the morphology or viability of small follicles, a sharp decrease in oocyte viability during in vitro growth was observed, presumably due to low-level expression of EpCAM on oocytes. The HER2-targeted DARPins had no detrimental effects on the morphology, viability, or in vitro growth of small follicles. HER2-positive breast cancer foci were fully eliminated from the ovarian cortex, and the reverse transcriptase polymerase chain reaction showed a decrease to basal levels of HER2 mRNA after purging.

Conclusion(s): Purging cancer metastases from ovarian cortex without impairing ovarian tissue integrity is possible by targeting tumor cell surface proteins with exotoxin A-fused DARPins. By adapting the target specificity of the cytotoxic DARPin fusions, it should be possible to eradicate metastases from all types of malignancies.

Keywords: DARPin-toxin fusion proteins; Oncofertility; ovarian cortex; purging; safety.