5548 Background: Efforts to improve immunotherapy for ovarian cancer (OC) have focused on optimizing T-cell effector-based strategies such as immune checkpoint inhibitors (ICIs), bispecific T-cell engagers (BiTEs), or chimeric antigen receptor (CAR)-T cells, or combining them with other therapeutic modalities. Relatively little is known about how adaptive resistance occurs or how to improve the intrinsic susceptibility of ovarian cancer cells to T cell-mediated cytolysis. Methods: An OC organoid derived from a patient who relapsed on immunotherapy (MGH538) was generated. OVCAR3 immune-edited cell line (OVCAR3 IE ) was generated by treating OVCAR3 wild-type (OVCAR3) tumor-bearing mice with activated T-cells, anti-PD-1, and anti-CTLA4 antibodies. Peritoneal tumors were then harvested and cultured in vitro . BiTEs-resistant cell line (OVCAR3 R ) was generated by treating OVCAR3 cells with anti-MUC16 BiTEs in vitro until the cells were resistant. Comparative RNA sequencing analysis was performed on OVCAR3, OVCAR3 IE , and OVCAR3 R . Protein expression was assessed by Western blot analysis. To evaluate cytotoxicity, OVCAR3, OVCAR3 IE , and OVCAR3 R were co-cultured with MUC16-specific CAR-T cells and MUC16-directed BiTEs, alone or in combination with pharmacologic inhibitors targeting EGFR, RAS, and Bcl-xL, including a Bcl-xL degrader DT2216. Results: MGH538, OVCAR3 IE and OVCAR3 R were confirmed to be resistant to T-cell directed cytolysis compared to OVCAR3. RNA sequencing analysis revealed significant differences in the transcriptome profiles of immune-naive and immunotherapy-exposed OC cells. In particular, significant changes in the RNA expression levels of three out of four ErbB family members (EGFR (ErbB1), ErbB2 and 3) were detected. In Western blot analysis, this finding was paralleled by significant changes in the ratio of phospho-ERK to ERK, an important component of ErbB downstream signaling. In functional assays, EGFR inhibition with gefitinib significantly enhanced CAR-T cell-mediated killing in OVCAR3, OVCAR3 IE and OVCAR3 R cells. Furthermore, blocking downstream EGFR signaling with a Ras inhibitor also restored sensitivity to immunotherapy. Finally, blocking anti-apoptotic signaling with Bcl-family inhibitor navitoclax, or the Bcl-xL inhibitor DT2216 restored susceptibility to both CAR-T cell and BiTEs-mediated killing. Conclusions: Adaptive resistance to T-cell mediated cytotoxicity appears to be mediated, at least in part, by upregulation of ErbB family members and consequent downstream Ras signaling and resistance to apoptosis. While further functional in vitro and therapeutic in vivo studies are warranted to investigate the translational potential of our findings, EGFR signaling may represent a promising target for clinical combination strategies.
Xu et al. (Wed,) studied this question.