The CD95 ligand (CD95L) is frequently overexpressed in cancers and tumor-associated endothelia, but also other immune cells like MDSCs and Tregs. Binding of CD95L expressed on tumors or regulatory immune cells to activated CD95-expressing effector cells triggers activation-induced apoptosis (AICD) or impairs their proliferation. In contrast, most tumors do also express CD95, but are intrinsically resistant to CD95-induced apoptosis. Thus, CD95L in the tumor microenvironment greatly contributes to the observed immunosuppression and escape from tumor surveillance by the immune system, making CD95 a potential immune checkpoint. Asunercept (APG101) is a fully human fusion protein consisting of the extracellular domain of CD95 and the Fc-domain of human IgG1, which efficiently inhibits CD95/CD95L signaling. Clinical efficacy has been demonstrated in a controlled randomized phase 2 study in patients with recurrent glioblastoma. Here we examined the effects of APG101 on innate and adaptive immune cells and subsequent effects on tumor cell killing. Subtypes of in vitro differentiated macrophages generated with and without exposure to APG101 were functionally and phenotypically analyzed by ELISA and multi-color flow cytometry following various stimuli. APG101 did not alter differentiation patterns and response of M1- and M2-like macrophages in vitro. Direct co-culture of monocytes with tumor cells resulted in an M2/TAM-like phenotype which was not influenced by APG101, but re-programming to an M1-like state was achieved by addition of a CD40 agonist. Effects of APG101 on the proliferation and activation of CD8+ T cells in the presence of autologous CD4+ T(reg) cells and allogeneic APC was assessed by CFSE-dilution and multi-color flow cytometry, respectively. The proliferation rate of CD8 T cells in co-cultures with CD4 T(reg) cells in response to stimulation with APCs was increased in the presence of APG101. Real-time cell analysis was performed employing direct co-cultures of activated T cells and tumor cell lines. Tumor killing assays using direct co-cultures of in vitro activated T cells with and without APG101 demonstrate that tumor killing was not impaired by APG101. Conclusion: Asunercept (APG101) is a potent inhibitor of pro-apoptotic/anti-proliferative CD95/CD95L signaling in immune cells and protects activated immune cells from activation induced cell death (AICD). Our results suggest that APG101 does not impair CD8 T cell activation, but rather supports their proliferation by disrupting CD95/CD95L interaction with regulatory T cells. Importantly, the primary anti-tumor killing mechanisms is most likely CD95L-independent and remains unaffected by the presence of APG101. The inhibition of CD95 signaling as an immune checkpoint represents an attractive and novel concept for immunologic treatment of tumors and the combination of Asunercept/APG101 with co-stimulatory TNFR-SF agonists is currently being investigated. Citation Format: Christian Merz, Jaromir Sykora, Rebecca Hussong, Meinolf Thiemann, Oliver Hill, Christian Gieffers. Neutralization of pro-apoptotic CD95L by Asunercept/APG101 does not impair anti-tumor immune responses [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4529.