To validate the relevance of those data inCYAD-01 cells from patients, we performedphenotypic analysis of CYAD-01 cellsproduced from AML patients. As illustratedin Figure 6, CYAD-01 cells from patientsexhibit a more variable phenotype comparedto healthy donors. Despite this variability,the CD4/CD8 ratio is also in favor of theCD8+ T-cells in AML CYAD-01 (Figure 6A).In addition, AML CYAD-01 cells are lesspositive for CD25+, while they do notexpress LAG-3, similarly to the healthydonor-derived CYAD-01 cells (Figure 6B andC respectively).Interestingly, the AML CYAD-01 memoryphenotype is less differentiated. Comparedto healthy donors, a reduction of effectormemory proportion (CD62L-CD45RA-) witha concomitant increase of the centralmemory population (CD62L+CD45RA-) isobserved (Figure 6D and E respectively).Concerning homing abilities and probablylinked to the memory phenotype, thechemokine receptor CXCR3 is almostabsent from AML CYAD-01. However, theCYAD-01 T-cell population positive forCXCR4 is enhanced. This is crucial for theAML indication, as it drives homing to thebone marrow, where the leukemia blasts andstem cells have been described to belocated [6].Finally, upon co-culture with K562 cells, bothhealthy donor and AML-derived CYAD-01cells secrete high level of IFN-γ. Importantly,this secretion was hindered using a NKG2Dspecific antibody, supporting a CAR-dependent secretion of IFN-γ (Figure 6H).

IL‐2, IL‐4, IL‐6 and IL‐17A

Uncovering the phenotype, functional and homing properties of NKG2D CAR T cells

Demoulin B, Breman E, Fontaine M, Huberty F, Daro D, Agaugue S, Sotiropoulou P, Gilham DE

B A C K G R O U N D

R E S U L T S

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 S

CYAD-01 cells generated using healthydonor or AML patient primary material,consist of an activated, non-exhausted,predominantly effector memory T cellpopulation. Interestingly, the strongexpression of CXCR4 may support theability of CYAD-01 cells to home to thebone marrow, an important property tofacilitate targeting of AML leukemicstem cells and blasts. The breadth ofcytokines produced by CYAD-01 cellsconfirms the ability of the NKG2D CARto provide strong activation and co-stimulatory signaling. These resultssupport the encouraging preliminaryresults from the THINK clinical trial.

F I G U R E 1 : N K G 2 D - C A R r e c o g n i z e s e i g h t d i f f e r e n t l i g a n d se x p r e s s e d i n a w i d e v a r i e t y o f c a n c e r s

Material and method :CYAD-01 Process: Healthy donors and AML THINK (NCT03018405) PBMCs were activated with OKT- 3 and IL-2 in X-Vivo15 for 2 days, then transduced using a retroviral vector coding for NKG2D-CAR for48hours. Transduced T cell were then cultured for 6 days in X-vivo15 in presence of NKG2D-blocking antibody and IL-2. At harvest, cells were washed then frozen until use.Flow cytometry: Thawed T cell were incubated with relevant antibodies for 30min at 4°C. After incubation, T cells were washed with PBS and analyzed on the Attune NxT flow cytometer. Data wereanalyzed using the FlowJo software.Cytokine release assay : Thawed CYAD-01 T cells were incubated with K562 at a cell ratio of 1:1 in X-vivo15 containing 5% FBS. After 24h incubation, supernatants were harvested and IFN-γ measured byELISA. Other cytokine were measured using a Multiplex assay.

T cells bearing a chimeric antigen receptor(CAR) consisting of the fusion of humanNKG2D with the intracellular domain ofCD3 (CYAD-01 CAR T cells) can recognizeeight stress ligands highly expressed inmany cancers [1,2] (Figure 1). Preliminaryresults from the THINK clinical trial(NCT03018405) showed complete response(morphologic leukemia-free state) in apatient with Acute Myeloid Leukemia (AML)upon CYAD-01 infusion [3]. To betterunderstand CYAD-01 T cells, we performeda thorough phenotypic and cytokineresponse characterization of CYAD-01 cellsafter cryopreservation. Importantly, wesought to compare CYAD-01 cells derivedfrom AML patients against those derivedfrom healthy donors to assess the impact oftumor. We also compared CYAD-01 cellsagainst control T cells expressing atruncated form of CD19 (tCD19) tounderstand the impact of the CAR on T cellphenotype.

F I G U R E 2 : C Y A D - 0 1 T c e l l s a r e C D 2 5 + , L A G - 3 - C D 8 +T c e l l s

F I G U R E 3 : C Y A D - 0 1 T c e l l s d i s p l a y a n e f f e c t o r m e m o r yp h e n o t y p e

Mock (1

0D)

CYAD-01

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F I G U R E 4 : C Y A D - 0 1 T c e l l s e x p r e s s C X C R 4 , C X C R 3 , C C R 3 a n d C C R 1 0

F I G U R E 5 : C Y A D - 0 1 T c e l l s s e c r e t e p r o - i n f l a m m a t o r yc y t o k i n e s u p o n c o c u l t u r e w i t h K 5 6 2 c a n c e r c e l l s .

F I G U R E 6 : C h a r a c t e r i z a t i o n o f C Y A D - 0 1 T c e l l s d e r i v e df r o m A M L p a t i e n t s

Mock (tC

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CYAD-01

% o

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8+

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Expression of at least one NKG2D ligand

Colorectal cancer 88%

Triple negative breast cancer 88%

Ovarian cancer 68%

Bladder cancer 78%

Pancreatic cancers 86%

NSCLC 92%

Health

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Flow cytometry data show that CYAD-01 Tcells generated from healthy donors areenriched in the CD8+ population comparedto control T cells (Figure 2A). Interestingly,an important proportion of CD4+ and CD8+T cells express high level of CD25. However,no LAG-3 (Figure 2C) or PD-1 (data notshown) signal could be identified at theCYAD-01 cell surface suggesting anactivated but non exhausted state of CYAD-01 T–cells.To determine whether the CYAD-01manufacturing process could affect T-celldifferentiation, the distinct memory T cellsubsets were analyzed, based on theexpression of CD62L and CD45RA (Figure3). The percentage of central memory T-cells (CD62L+ CD45RA-) was reduced whenT-cells are transduced with NKG2D-CAR.Alongside, effector memory T cells (CD62L-CD45RA-) is enriched.The screening of chemokine receptors ofhealthy donor-derived CYAD-01 T cellsshows an important T cell populationpositive for CXCR4 (bone marrow and lymphnode homing [4] ; Figure 4A) and CXCR3(inflammation site homing [4]; Figure 4B).Interestingly, a fraction of CYAD-01 T-cellsexpresses CCR3 (Figure 4C) and themajority of CD4+ T cells are positive forCCR10 (Figure 4D).Multiplex bead analysis of healthy donor-derived CYAD-01 cells revealed secretion ofa panel of cytokines, including IFN-γ (Figure5A), IL-2, IL-4, IL-6, and IL-17A (Figure 5B)indicating the establishment of pro-inflammatory microenvironment, potentiallytriggering the host immune response, aspreviously shown in animal models [5].

A

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)

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Adapted from Fernandez-Messina L. et al.

Agaugue et al. ESMO 2018 poster 1179P

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

4. Mora and von Adrian, 2006. TRENDS in Immunology5. Demoulin B. et al. 2018 Future oncology.6. Ishikawa et al. 2007. Nature Biotechnology

REFERENCES:1. Sentman and Meehan. 2014, Cancer J.2. Fernandez-Messina L. et al. 2012 Frontiers in Immunology3. Sallman et al. 2018 Haematologica