Auristatin F-Delivering SNAP-Tag Based Recombinant Antibody-Drug Conjugate Targeting EpCAM Demonstrating Selective In vitro Killing of Cervical and Triple-Negative Breast Cancer Cell Lines

Author(s): Ursula-Claire Andong-Koung-Edzidzi , Thabo Matshoba , Dirk Lang , Roger Hunter and Stefan Barth

Background: Triple-negative breast cancer is an aggressive subtype of breast cancer lacking the ER, PR, and HER2 receptor. Hence, standard treatment is limited. Cervical cancer is considered a preventable cancer since it often occurs secondary to long-term high-risk HPV infection. Vaccines against the virus are available and globally have resulted in reduced incidence and mortality yet, in some regions cervical cancer remains the second cause of cancer related mortality in women, second only to breast cancer. Therefore, the importance of a single effective and specific treatment against both cancers could not be understated.

Methodology: scFv-SNAP-tag based fusion protein was transiently expressed in a mammalian expression system, and the secreted proteins were isolated from cell culture supernatants by immobilized metal affinity chromatography. Subsequently, auristatin F (AURIF) anticancer drug was conjugated to the scFv-SNAP to assess the dose-dependent activities of the therapeutic protein on EpCAM-positive TNBC and cervical cancer cell lines.

Results: The surface binding of the fluorescently labelled SNAP-tag based fusion protein was observed across all cell lines; however, internalization was seen on one EpCAM-positive TNBC and all cervical cancer cell lines. The ?EpCAM(scFv)-SNAP-linker-AURIF ADC demonstrated dose-dependent killing on target cell lines.

Conclusion: Fluorophore labelled EpCAM(scFv)-SNAP works well in flow cytometry and fluorescent microscopy, and can be further exploited as a diagnostic tool. The ADC demonstrated selective killing of highly EpCAM-positive cell lines. This might have further implications for future human application as a prescreening of patients might allow to identify the ones with higher expression levels benefitting from such an EpCAM targeting immunotherapy.