Transfection of various Fas constructs into the Hey-A8 Fas KO tumor cell lines was achieved by jetOPTIMUS for recombinant Fas (wildtype or mutated) cloned in pCDN3.1 vector. 60–70% of confluent cells were grown in a 10 cm culture dish. 10 µg DNA was diluted into 1000 µL jetOPTIMUS buffer and vortexed. This was followed by the addition of 10 µL jetOPTIMUS into the DNA solution (ratio 1:1 corresponding to µg DNA: µL reagent) and vortexed and spun down briefly. The mixture was incubated for 10 min at room temperature. Next, the transfection mix was added dropwise onto the cells in a serum-containing medium and distributed evenly. Plates were incubated at 37 °C for 24 h. The next day transfection medium was replaced with by cell growth medium and cells were allowed to grow for another day before starting G418 (2 mg/ml) selection. Media was changed every day, and a reliable GFP signal was evident 72 h of transfection. For long-term stable line generation, lentiviral method was used as described in the next section.

Receptor clustering is the most critical step to activate extrinsic apoptosis by death receptors belonging to the TNF superfamily. Although clinically unsuccessful, using agonist antibodies, the death receptors-5 remains extensively studied from a cancer therapeutics perspective. However, despite its regulatory role and elevated function in ovarian and other solid tumors, another tumor-enriched death receptor called Fas (CD95) remained undervalued in cancer immunotherapy until recently, when its role in off-target tumor killing by CAR-T therapies was imperative. By comprehensively analyzing structure studies in the context of the binding epitope of FasL and various preclinical Fas agonist antibodies, we characterize a highly significant patch of positively charged residue epitope (PPCR) in its cysteine-rich domain 2 of Fas. PPCR engagement is indispensable for superior Fas agonist signaling and CAR-T bystander function in ovarian tumor models. A single-point mutation in FasL or Fas that interferes with the PPCR engagement inhibited apoptotic signaling in tumor cells and T cells. Furthermore, considering that clinical and immunological features of the autoimmune lymphoproliferative syndrome (ALPS) are directly attributed to homozygous mutations in FasL, we reveal differential mechanistic details of FasL/Fas clustering at the PPCR interface compared to described ALPS mutations. As Fas-mediated bystander killing remains vital to the success of CAR-T therapies in tumors, our findings highlight the therapeutic analytical design for potentially effective Fas-targeting strategies using death agonism to improve cancer immunotherapy in ovarian and other solid tumors.