CYAD-101: a first non-gene edited allogeneic CAR T cell Despite the variation in phenotype between donors, the inhibition of alloreactivity of CYAD-101 was confirmed in vitro by a sharp reduction in IFN-γ production upon TCR-mediated activation with 200ng/ml of OKT3 (Figure 4A) and in vivo by inhibition of GVHD (Figure 4B). NSG mice receiving mock T cells succumbed to xenoGvHD with a median survival of 49 days, while all mice injected with CYAD-101 cells were alive at the end of the experiment (day 56). CYAD-101 can effectively target NKG2D ligand-expressing tumor cell lines To assess the in vitro functionality of NKG2D CAR in a TIM context, CYAD-101 cells were co-cultured with the NKG2D- ligand positive human Chronic Myeloid Leukemia (CML) cell line K562 in the presence or absence of anti-NKG2D mAb (5μg/mL). After 24 hours, IFN-γ release, reflecting activation of T cells through the NKG2D-CAR, was analyzed by ELISA. The results illustrated in the left panel of Figure 5A show that CYAD-101 cells produced high amounts of IFNγ upon coculture with K562 cells while this production was completely inhibited in the presence of the anti-NKG2D blocking mAb, demonstrating the specificity of the recognition of NKG2D ligands by the NKG2D-CAR expressed by CYAD-101 cells. The kinetics of CYAD-101 cytolytic activity was further assessed by coculture with PANC-1 human pancreatic cancer cells (Figure 5A, right panel). Results from a 72-hour culture showed 80% reduction of living PANC-1 cells. Importantly, anti-tumor activity of CYAD-101 was confirmed in vivo using an orthotopic mouse model of colorectal cancer. Three weekly injections of CYAD- 101 cells significantly delayed tumor growth and increased survival of more than two weeks without evidence of GvHD (Figure 5B). CYAD-101: an allogeneic NKG2D CAR T cell therapy using a TCR inhibitory molecule A. Michaux, E. Breman, S. Mauën, D. Gilham, S. Agaugué ABSTRACT Current Chimeric Antigen Receptor (CAR) T cell therapies rely mostly on patient’s autologous blood cells leading to challenges resulting from the variability of the starting material and the time pressure for manufacturing. Use of allogeneic T cells derived from a healthy donor can circumvent these issues. However, one limitation of allogeneic T cell use is the potential to induce life-threatening graft versus host disease (GvHD), triggered by the recognition of foreign human leukocyte antigen (HLA) molecules expressed on the patient’s cells by the T Cell Receptor (TCR) of the lymphocytes of the donor. To avoid GvHD, we inhibited TCR signaling using a non-gene editing technology. We co- expressed together with a NKG2D-based CAR a TCR inhibitory molecule (TIM) composed of a truncated form of CD3ζ, termed CYAD-101 cells (Figure 1A). The CAR composed of the ITAM-bearing signaling cytoplasmic domain of human CD3ζ fused with the full-length NKG2D allows signaling upon binding to NKG2D ligands expressed on tumor cells, triggering cancer cell killing, while the TIM- incorporated TCR lacks a signaling moiety and cannot signal, thus avoiding alloreactivity (Figure 1B, C). RESULTS TIM incorporates into the TCR complex To confirm the ability of TIM to incorporate TCR complex, PBMCs from 5 different donors were transduced with shRNA against CD3ζ together or not with TIM construct. As expected, T cells transduced with shRNA alone showed a strong decrease in TCR cell surface expression (Figure 2A). However, TIM co- expression partially restored TCR expression at cell surface of T cell implying that the TIM incorporates into the TCR complex and compensates for the loss of CD3ζ. In addition, CD3ζ phosphorylation monitored by FACS was impaired in TIM transduced T cells when compared to control cells upon 5 min TCR activation with anti-CD3 antibody (Figure 2B). CYAD-101 characterization CYAD-101 cells were harvested and cryopreserved. Phenotypic evaluation (Figure 3) of CYAD-101 cells produced from five different healthy donors revealed variability in CD4/CD8 ratio and in percentage of central and effector memory populations related to the donor variability. Importantly, CYAD-101 cells were mainly composed of a non-activated and non-exhausted population (i.e. CD25- /CD69- and PD1-/LAG3-). RESULTS FIGURES & TABLES CONCLUSION and FUTURE DEVELOPMENT In this study, we characterized CYAD-101 CAR T cells in vitro and in vivo. We demonstrated that these allogeneic non-gene edited, NKG2D-based CAR T cells lack alloreactivity and display effective anti-cancer activity. A phase I clinical trial will be initiated in 2018 to assess the safety and clinical activity of CYAD-101 CAR T cells in patients with unresectable metastatic colorectal cancer (Figure 6). FIGURE 1:CYAD-101: an allogeneic CAR T cell co-expressing TIM and a NKG2D-based CAR AFFILIATIONS: *Research & Development department, Celyad SA, Mont-Saint-Guibert, Belgium RELEVANT LITERATURE: . A . B. FIGURE 2: TIM incorporate TCR complex impairing its activation FIGURE 3: CYAD-101 characterization FIGURE 4:TIM impairs CYAD-101 alloreactivity in vitro and in vivo CYAD-101 NSG mice FIGURE 5:CYAD-101 is potent against tumors expressing NKG2D ligands in vitro and in vivo A. In vitro TCR activation B. In vivo xenoGvHD model A. In vitro cytotoxicity assay B. In vivo orthotopic colorectal cancer model 5 10 15 20 25 30 35 0 5 10 10 1 10 11 1.5 10 11 Days post infusion Bioluminescence (photons / sec / mm 2 ) CTR (tCD19) CYAD-101 *** CTR (tCD19) CYAD-101 FIGURE 6:CYAD-101 in the clinic 10. 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