CYAD-101: a first non-gene editedallogeneic CAR T cell

Despite the variation in phenotypebetween donors, the inhibition ofalloreactivity of CYAD-101 was confirmedin vitro by a sharp reduction in IFN-γproduction upon TCR-mediated activationwith 200ng/ml of OKT3 (Figure 4A) and invivo by inhibition of GVHD (Figure 4B).NSG mice receiving mock T cellssuccumbed to xenoGvHD with a mediansurvival of 49 days, while all mice injectedwith CYAD-101 cells were alive at the endof the experiment (day 56).

CYAD-101 can effectively target NKG2Dligand-expressing tumor cell lines

To assess the in vitro functionality ofNKG2D CAR in a TIM context, CYAD-101cells were co-cultured with the NKG2D-ligand positive human Chronic MyeloidLeukemia (CML) cell line K562 in thepresence or absence of anti-NKG2D mAb(5μg/mL). After 24 hours, IFN-γ release,reflecting activation of T cells through theNKG2D-CAR, was analyzed by ELISA. Theresults illustrated in the left panel of Figure5A show that CYAD-101 cells producedhigh amounts of IFNγ upon coculture withK562 cells while this production wascompletely inhibited in the presence of theanti-NKG2D blocking mAb, demonstratingthe specificity of the recognition ofNKG2D ligands by the NKG2D-CARexpressed by CYAD-101 cells. The kineticsof CYAD-101 cytolytic activity was furtherassessed by coculture with PANC-1human pancreatic cancer cells (Figure 5A,right panel). Results from a 72-hour cultureshowed 80% reduction of living PANC-1cells. Importantly, anti-tumor activity ofCYAD-101 was confirmed in vivo using anorthotopic mouse model of colorectalcancer. Three weekly injections of CYAD-101 cells significantly delayed tumorgrowth and increased survival of morethan 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

A B S T R A C T

Current Chimeric Antigen Receptor (CAR)T cell therapies rely mostly on patient’sautologous blood cells leading tochallenges resulting from the variability ofthe starting material and the time pressurefor manufacturing. Use of allogeneic T cellsderived from a healthy donor cancircumvent these issues. However, onelimitation of allogeneic T cell use is thepotential to induce life-threatening graftversus host disease (GvHD), triggered bythe recognition of foreign human leukocyteantigen (HLA) molecules expressed on thepatient’s cells by the T Cell Receptor (TCR)of the lymphocytes of the donor. To avoidGvHD, we inhibited TCR signaling using anon-gene editing technology. We co-expressed together with a NKG2D-basedCAR a TCR inhibitory molecule (TIM)composed of a truncated form of CD3ζ,termed CYAD-101 cells (Figure 1A). TheCAR composed of the ITAM-bearingsignaling cytoplasmic domain of humanCD3ζ fused with the full-length NKG2Dallows signaling upon binding to NKG2Dligands expressed on tumor cells,triggering cancer cell killing, while the TIM-incorporated TCR lacks a signaling moietyand cannot signal, thus avoidingalloreactivity (Figure 1B, C).

R E S U L T S

TIM incorporates into the TCR complexTo confirm the ability of TIM toincorporate TCR complex, PBMCs from 5different donors were transduced withshRNA against CD3ζ together or not withTIM construct. As expected, T cellstransduced with shRNA alone showed astrong decrease in TCR cell surfaceexpression (Figure 2A). However, TIM co-expression partially restored TCRexpression at cell surface of T cell implyingthat the TIM incorporates into the TCRcomplex and compensates for the loss ofCD3ζ. In addition, CD3ζ phosphorylationmonitored by FACS was impaired in TIMtransduced T cells when compared tocontrol cells upon 5 min TCR activationwith anti-CD3 antibody (Figure 2B).CYAD-101 characterizationCYAD-101 cells were harvested andcryopreserved. Phenotypic evaluation(Figure 3) of CYAD-101 cells producedfrom five different healthy donors revealedvariability in CD4/CD8 ratio and inpercentage of central and effectormemory populations related to the donorvariability. Importantly, CYAD-101 cellswere mainly composed of a non-activatedand non-exhausted population (i.e. CD25-/CD69- and PD1-/LAG3-).

R E S U L T SF I G U R E S & T A B L E S

C O N C L U S I O N a n d F U T U R E D E V E L O P M E N T

In this study, we characterizedCYAD-101 CAR T cells in vitro and invivo. We demonstrated that theseallogeneic non-gene edited,NKG2D-based CAR T cells lackalloreactivity and display effectiveanti-cancer activity. A phase I clinicaltrial will be initiated in 2018 to assessthe safety and clinical activity ofCYAD-101 CAR T cells in patientswith unresectable metastaticcolorectal cancer (Figure 6).

F I G U R E 1 : C Y A D - 1 0 1 : a n a l l o g e n e i c C A R T c e l lc o - e x p r e s s i n g T I M a n d a N K G 2 D - b a s e d C A R

AFFILIATIONS: *Research & Development department, Celyad SA, Mont-Saint-Guibert, Belgium

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13. Barber A, Zhang T, DeMars LR, Conejo-Garcia J, Roby KF, Sentman CL. Chimeric NKG2D Receptor-Bearing T Cells as Immunotherapy forOvarian Cancer. Cancer Res 2007; 67:5003–8.

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1. Fesnak AD, June CH, Levine BL. Engineered T cells: the promise and challenges of cancer immunotherapy. Nat Rev Cancer 2016; 16:566–81.2. Rossig C. CAR T cell immunotherapy in hematology and beyond. Clin Immunol 2017 [cited 2018 Feb 9];3. Brenner MK. Next Steps in the CAR Journey of a Thousand Miles. Mol Ther 2017; 25:2226–7.4. Sentman CL, Meehan KR. NKG2D CARs as cell therapy for cancer. Cancer J 2014; 20:156–98. Lanier LL. NKG2D Receptor and Its Ligands in Host Defense. Cancer Immunol Res 2015; 3:575–82..9. Spear P, Wu M-R, Sentman M-L, Sentman CL. NKG2D ligands as therapeutic targets. Cancer Immun 2013;13:8.

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