Owing to the successful employment of cisplatin-based chemotherapy, germ cell tumors (GCTs) have achieved high curability rates. Nonetheless, acquired refractoriness to cisplatin and tumor recurrence remain a clinical challenge, with a current lack of therapeutic options for these patients. With the recent surge in exploration of the cancer surfaceome and target antigens, antibody-drug conjugates (ADCs) have become a feasible anti-tumor modality in various cancer types. In our study, we aimed to broaden the scope of the ADC approach to cisplatin-refractory GCTs, with mirvetuximab soravtansine (MIRV) targeting FRα and trastuzumab deruxtecan (T-Dxd) targeting HER2.

A panel of 29 parental and cisplatin-resistant (CisR) isogenic variants of GCT cell lines was subjected to mRNA gene expression analysis of target genes encoding FRα and HER2 (FOLR1 and ERBB2, respectively) with RT-qPCR. Treatment sensitivity of chemoresistant GCT cell lines to MIRV and T-Dxd was assessed with kinetic proliferation assays (IncuCyte Live Cell Imaging or Axion impedance assay) and with an endpoint luminescence assay on day 3 after treatment.

Gene expression analysis revealed increased FOLR1 levels and substantial ERBB2 levels in the majority of cell lines. Furthermore, both genes were upregulated in several resistant variants compared to their parental counterparts, with notable differences observed in ERBB2 expression. Potent anti-proliferative effects of MIRV were noted in resistant variants of seminoma (TCam2 CisR) and choriocarcinoma (JAR CisR, JEG-3 CisR) GCT subtypes, which correlated with higher FOLR1 levels. Similarly, the highest cytotoxic effect of T-Dxd was observed in cancer cells derived from testicular yolk sac tumor (NCR-G1) and in the resistant variant of embryonal carcinoma (NEC-8 CisR), both of which had among the highest ERBB2 expression levels. Interestingly, some chemoresistant variants with high ERBB2 expression, such as Tera-2 CisR or NOY-1 CisR, did not yield the expected response to T-Dxd. This may have occurred due to the lack of efficacy of the conjugated payload, deruxtecan, or to alterations or heterogeneity of the surface protein expression in these cells.

Our results indicate the preclinical potential of targeting FRα/HER2 with ADC-mediated therapy in GCTs, particularly within the chemorefractory disease setting. Further research and validation of this approach in vivo is warranted.

This work was financially supported by projects APVV-24-0168; VEGA 2/0059/25; the Slovak Cancer Research Foundation and the League Against Cancer, Slovakia.