CYAD-101: an allogeneic NKG2D CAR T cell therapy using a ......CYAD-101: a first non-gene edited...
1
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. Zhang T, Barber A, Sentman CL. Generation of Antitumor Responses by Genetic Modification of Primary Human T Cells with a Chimeric NKG2D Receptor. Cancer Res 2006; 66:5927–33. 11. Barber A, Rynda A, Sentman CL. Chimeric NKG2D expressing T cells eliminate immunosuppression and activate immunity within the ovarian tumor microenvironment. J Immunol2009; 183:6939–47. 12. Zhang T, Barber A, Sentman CL. Chimeric NKG2D Modified T Cells Inhibit Systemic T-Cell Lymphoma Growth in a Manner Involving Multiple Cytokines and Cytotoxic Pathways. Cancer Res 2007; 67:11029–36. 13. Barber A, Zhang T, DeMars LR, Conejo-Garcia J, Roby KF, Sentman CL. Chimeric NKG2D Receptor-Bearing T Cells as Immunotherapy for Ovarian Cancer. Cancer Res 2007; 67:5003–8. 14. Demoulin B, Cook WJ, Murad J, Graber DJ, Sentman M-L, Lonez C, Gilham DE, Sentman CL, Agaugue S. Exploiting natural killer group 2D receptors for CAR T-cell therapy. Futur Oncol 2017; 13:1593–605. 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; 20:156–9 8. 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. C. IFN- (ng/ml) Percent survival 0 10 20 30 40 50 0 50 100 Days after injection Percent survival CTR (tCD19) CYAD-101 **** D30 OKT3 A . B .
Transcript of CYAD-101: an allogeneic NKG2D CAR T cell therapy using a ......CYAD-101: a first non-gene edited...
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
RELEVANT LITERATURE: .
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F I G U R E 2 : T I M i n c o r p o r a t e T C R c o m p l e x i m p a i r i n gi t s a c t i v a t i o n
F I G U R E 3 : C Y A D - 1 0 1 c h a r a c t e r i z a t i o n
F I G U R E 4 : T I M i m p a i r s C Y A D - 1 0 1 a l l o r e a c t i v i t y i n v i t r oa n d i n v i v o
CYAD-101
NSG mice
F I G U R E 5 : C Y A D - 1 0 1 i s p o t e n t a g a i n s t t u m o r s e x p r e s s i n gN K G 2 D l i g a n d s i n v i t r o a n d i n v i v o
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 30 350
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F I G U R E 6 : C Y A D - 1 0 1 i n t h e c l i n i c
10. Zhang T, Barber A, Sentman CL. Generation of Antitumor Responses by Genetic Modification of Primary Human T Cells with a ChimericNKG2D Receptor. Cancer Res 2006; 66:5927–33.
11. Barber A, Rynda A, Sentman CL. Chimeric NKG2D expressing T cells eliminate immunosuppression and activate immunity within the ovariantumor microenvironment. J Immunol2009; 183:6939–47.
12. Zhang T, Barber A, Sentman CL. Chimeric NKG2D Modified T Cells Inhibit Systemic T-Cell Lymphoma Growth in a Manner Involving MultipleCytokines and Cytotoxic Pathways. Cancer Res 2007; 67:11029–36.
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.
14. Demoulin B, Cook WJ, Murad J, Graber DJ, Sentman M-L, Lonez C, Gilham DE, Sentman CL, Agaugue S. Exploiting natural killer group 2Dreceptors for CAR T-cell therapy. Futur Oncol 2017; 13:1593–605.
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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