Mondal, TanmoyGaur, HimanshuWamba, Brice E. N.Michalak, Abby GraceStout, CamrynWatson, Matthew R.Aleixo, Sophia L.Singh, ArjunCondello, SalvatoreFaller, RolandLeiserowitz, Gary ScottBhatnagar, SanchitaTushir-Singh, Jogender2024-03-262024-03-262023Mondal T, Gaur H, Wamba BEN, et al. Characterizing the regulatory Fas (CD95) epitope critical for agonist antibody targeting and CAR-T bystander function in ovarian cancer. Cell Death Differ. 2023;30(11):2408-2431. doi:10.1038/s41418-023-01229-7https://hdl.handle.net/1805/39498Receptor 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.en-USAttribution 4.0 InternationalTumour heterogeneityPreclinical researchCancer microenvironmentArticle