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Blockade of CD95/CD95L Death Signaling Enhances CAR T Cell Persistence and Antitumor Efficacy

Introduction
 Despite the encouraging outcome of anti-CD19 chimeric antigen receptor T (CAR T) cell therapy in patients with B cell malignancies, CAR T cell persistence remains a major clinical challenge. Activation-induced cell death (AICD) is a programmed cell death caused by the interaction of CD95 and CD95L. Through specific blocking of the CD95-CD95L pathway, the CD95L inhibitor APG101 (Asunercept, Apogenix AG, Heidelberg) could prevent activated T cells from AICD. APG101 is a fully human fusion protein consisting of the extracellular domain of CD95 receptor and the Fc domain of an IgG antibody. Thus, we evaluated whether a blockade of the CD95L pathway through APG101 might improve CAR T cell persistence and enhance antitumor efficacy.
 Methods
 Human peripheral blood mononuclear cells (PBMCs) from healthy donors were stimulated by plate-bound CD3 and CD28 antibodies, and thereafter transduced with a 3rd generation CD19.CAR.CD28.CD137zeta retroviral vector. An in vitro co-culture stress test assay was employed to assess the functional status and viability of CD19.CAR T cells upon repetitive stimulation with CD19+ tumor cells, i.e. Daudi cells. CAR T cells (5.0 x 105 per well) were co-cultured with tumor cells at a 1:1 E:T ratio (round I) in the presence of APG101. Additional tumor cells were supplied to the co-culture every 24 hours. After 3 rounds (72 hr) of stimulation, tumor cells (CD3-CD19+) and CAR T cells (CD3+CD19-) were harvested for FACS analysis. To assess the antigen-induced CAR T cell proliferation, CAR T cells were preloaded with Cell Trace Violet cytosolic dye and cocultured with tumor cells for 72 hours.
 Results
 Activation-induced cell death of CAR T cells was observed after repeated antigenic stimulation, accompanied by increased CD95L expression. CD4+ CAR T cells were more susceptible to AICD compared to CD8+ CAR T cells. But, there was no difference in the expression of CD95L between CD4+ and CD8+ CAR T cells. Interestingly, addition of APG101 significantly inhibited CD95L expression and resulted in a lower level of CAR T cell death. Importantly, APG101 did not hamper the activation and proliferation of CAR T cells but was able to restore CAR T cell viability. The expression of PD1, TIM3 and LAG3 were also up-regulated after successive stimulation, however, their expression on CAR T cells were not influenced by APG101. After 3 days of co-culture, the number of CAR T cells increased in the presence of APG101 (7.9 x 105 vs6.0 x 105, P = 0.01) and residual tumor cells were diminished (1.7 x 105 vs2.7 x 105, P = 0.02). Of note, APG101 itself did not affect CAR T cells or tumor cells when cultured separately. Moreover, the central memory CAR T (TCM) cell subset showed higher CD95L expression after coculturing which could be inhibited by APG101. Therefore, the addition of APG101 to the coculture resulted in a significant accumulation of TCM subset after APG101 treatment.
 Conclusion
 Upregulation of CD95L after repeated antigen stimulation was reversed by APG101. CD95L blockade enhanced CAR T cell survival and promoted killing of tumor cells in vitro. Combining CAR T cell therapy with CD95L inhibitor might improve CAR T cell persistence in vivo and thus enhance the effect of CAR T cell therapy.
 
 
 Schmitt: Therakos Mallinckrodt: Other: Financial Support . Kneisel:Apogenix AG: Employment. Hoeger:Apogenix AG: Employment, Membership on an entity's Board of Directors or advisory committees. Schmitt:MSD: Membership on an entity's Board of Directors or advisory committees, Other: Sponsoring of Symposia; Therakos Mallinckrodt: Other: Financial Support.
