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NKG2D-based chimeric antigen receptor therapy induced remission in a relapsed/refractory acute myeloid leukemia patient
by David A. Sallman, Jason Brayer, Elizabeth M. Sagatys, Caroline Lonez, Eytan Breman, Sophie Agaugué, Bikash Verma, David E. Gilham, Frédéric F. Lehmann, and Marco L. Davila
Haematologica 2018 [Epub ahead of print]
Citation: David A. Sallman, Jason Brayer, Elizabeth M. Sagatys, Caroline Lonez, Eytan Breman, Sophie Agaugué, Bikash Verma, David E. Gilham, Frédéric F. Lehmann, and Marco L. Davila. NKG2D-based chimeric antigen receptor therapy induced remission in a relapsed/refractory acute myeloidleukemia patient. Haematologica. 2018; 103:xxxdoi:10.3324/haematol.2017.186742
Publisher's Disclaimer.E-publishing ahead of print is increasingly important for the rapid dissemination of science.Haematologica is, therefore, E-publishing PDF files of an early version of manuscripts thathave completed a regular peer review and have been accepted for publication. E-publishingof this PDF file has been approved by the authors. After having E-published Ahead of Print,manuscripts will then undergo technical and English editing, typesetting, proof correction andbe presented for the authors' final approval; the final version of the manuscript will thenappear in print on a regular issue of the journal. All legal disclaimers that apply to thejournal also pertain to this production process.
Copyright 2018 Ferrata Storti Foundation.Published Ahead of Print on April 27, 2018, as doi:10.3324/haematol.2017.186742.
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NKG2D-based chimeric antigen receptor therapy induced remission in a
relapsed/refractory acute myeloid leukemia patient.
Authors and affiliations
David A. Sallman1*, Jason Brayer1*, Elizabeth M. Sagatys2, Caroline Lonez3, Eytan Breman3,
Sophie Agaugué3, Bikash Verma4, David E. Gilham3, Frédéric F. Lehmann3, Marco L Davila5
1Malignant Hematology and 2Hematopathology and Laboratory Medicine, H. Lee Moffitt
Cancer Center and Research Institute, Tampa, FL, USA
3 Celyad, SA, Mont-Saint-Guibert, Belgium
4 Celyad, SA, New York, NY, USA
5Blood & Marrow Transplantation and Cellular Immunotherapy, H. Lee Moffitt Cancer
Center and Research Institute, Tampa, FL, USA
*These authors contributed equally to this work.
Contact information for correspondence: David A. Sallman or Marco L Davila, H. Lee
Moffitt Cancer Center, Tampa, FL, USA. Phone: 1-888-663-3488, Email:
David.Sallman@moffitt.org or Marco.Davila@moffitt.org
Running heads: Remission in an r/r AML patient with a CAR-T
Word count for the main text: 1286
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Acute Myeloid Leukemia (AML) is the most common acute leukemia affecting adults
characterized by the accumulation of immature myeloblasts in the marrow or peripheral
blood. Natural Killer Group 2D (NKG2D) is an activating receptor expressed on Natural
Killer (NK) cells and activated CD8+ T cells, which triggers cytotoxicity following
recognition of its ligands (namely MHC class I polypeptide-related sequence A and B
(MICA/B) and unique long protein 16 (UL-16) binding protein 1 to 6 (ULBP1 to 6)) at the
surface of stressed, infected and most notably transformed cells 1. Consequently, a broad
range of primary tumors express NKG2D ligands (NKG2DL) and can be targeted by
NKG2D-based immunotherapies 2, 3. NKG2DL expression was reported in AML, with data
reporting 67% to 100% expression in MICA/B and/or ULBP2-3 expression in blasts 4, 5 with
all blasts expressing at least one ligand [Dulphy N, Toubert A et al., unpublished data].
CYAD-01 are autologous T-cells genetically modified to express a chimeric antigen
receptor (CAR) comprising a fusion of the human full-length NKG2D receptor with the CD3ζ
signaling domain 6. Although the co-stimulatory molecule DNAX-activating protein 10
(DAP10) is not part of the transgene, NKG2D associates with this molecule for membrane
stabilization to provide the secondary activation signal 1. The NKG2D-CAR construct binds 8
different ligands in an MHC-independent fashion expressed by cancer cells of diverse origins
3, 7, 8. Because the ligands for the NKG2D receptor are absent or expressed at very low levels
in normal tissues 1, 9, specificity for tumors is considered to be high. In agreement, we
previously demonstrated no recognition of normal peripheral blood mononuclear cells nor
healthy bone marrow from healthy patients by CYAD-01 in vitro 10. Preclinical studies have
shown that CYAD-01 mediates potent anti-tumor activity against both hematological and
solid tumors without the requirement of prior lymphodepleting chemotherapy 3, 6. Single
intravenous administration of low dose autologous CYAD-01 (maximum 3x107 flat dose),
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without prior chemotherapy, has been tested in a completed Phase I study (NCT02203825)
which evaluated 12 patients with relapsed/refractory AML (r/r AML), myelodysplastic
syndrome (MDS) or multiple myeloma (MM) 11. The preliminary data obtained suggested
encouraging signs of activity with one AML patient treated at the 3x107 CYAD-01 dose-level
having hematologic improvement for three months post-CYAD-01 treatment 11. Building on
these initial results and preclinical data supporting multiple CYAD-01 infusions are necessary
for disease eradication, we initiated the THINK study (ClinicalTrials.gov number:
NCT03018405) evaluating the safety and clinical activity of multiple treatment
administrations of CYAD-01 in different solid and hematological indications 6.
We report here the case of a 52 years old male with r/r AML. The patient had
+8/del(7)(q22q36), FLT3/NPM1 wild-type AML that was primary refractory to induction
with 7+3 with daunorubicin and cytarabine. The patient subsequently received salvage
chemotherapy with cladribine, cytarabine, G-CSF and mitoxantrone (CLAG-M) and achieved
CR1 followed by two cycles of CLA consolidation. Following a 7 months remission where
allogeneic hematopoietic stem cell transplantation (allo-HSCT) was delayed to allow for
pulmonary function test recovery, the patient had recurrent cytopenias and bone marrow
(BM) biopsy confirmed relapsed disease with hypocellularity, 7% blasts in numerous clusters
and dysmegakaryopoiesis (Fig. 1A and 1B). The patient developed severe constitutional
symptoms with fatigue, anorexia, body pain and drenching night sweats. Given no standard
treatment options, the patient enrolled in the THINK trial and underwent apheresis followed
by CYAD-01 infusions at the initial dose level of 3x108 cells (flat dose)/injection every 2
weeks for 3 administrations without any lymphodepleting chemotherapy 6. CYAD-01 was
well tolerated with non-related grade 1 adverse events without cytokine release syndrome
(CRS) or neurotoxic effects. BM evaluation at day +28 following 2 CYAD-01 infusions
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showed morphologic leukemia-free state (MLFS, 2% blasts) with normocellularity and
trilineage hematopoiesis, which was confirmed at day +56 BM with del(7q) by FISH at 7%
(Fig. 1C). Serial next-generation sequencing (NGS) of BM-MNCs showed persistent
DNMT3A R882H mutation, but also identified a new IDH2 R172K mutation. The patient had
resolution of symptoms with improved hematopoiesis, which continued to improve until the
patient was treated with an allo-HSCT on day +97 post CYAD-01 (Fig. 1D). At disease
evaluation 100 days post allo-HSCT (day +197 post CYAD-01), the patient achieved a
complete molecular remission by serial NGS with 100% donor chimerism (Fig. 1D). To date,
the patient has maintained a CR 6 months from allo-HSCT and 9 months following initial
CYAD-01 infusion. Fig. 2A shows timeline of the patient’s history of treatments and
responses.
Molecular analysis indicated a low level of CYAD-01 cells in the periphery until day +4 post
injection. The patient CYAD-01 product primarily consisted of effector-memory phenotype
CD8+ T-cells with robust target cell-directed cytotoxicity and interferon (IFN)-γ secretion.
(Fig. S1A and S1B). Immunohistochemistry (IHC) analysis on the baseline BM taken showed
positive staining of foci of neoplastic cells for all NKG2DL assessed (Fig S2), with the
strongest expression for MICA/MICB (Fig. 2B). Of note, there was no NKG2DL expression
on the non-neoplastic BM cells. Finally, it is important to mention no detectable level of the
transgene was evidenced at day +1, +4 and +32 post-injection, using a Vector copy number
(VCN) qPCR-based assay, suggesting poor in vivo expansion and persistence of the injected
cell product.
CAR therapy has brought forth a paradigm change in the management of refractory chemo-
resistant B-cell leukemia 12. However, the choice of target in AML has been challenging due
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to their normal expression on hematopoietic stem cells leading to potential irreversible
hematopoietic toxicity 13. Thus, current CAR-T strategies in AML either require a backup
allo-HSCT or unproven shut-off mechanisms 14. In contrast, NKG2DL are upregulated in
transformed cells with minimal expression in normal tissues 1. This was confirmed by the
absence of significant adverse events of CYAD-01 in this AML patient and no Grade 3 or
above adverse events related to treatment in any of the AML patients enrolled in the THINK
trial up to now. This is of critical clinical importance given CD19 specific CAR-T cell
therapies with grade 5 toxicities including CRS, neurotoxicity and on-target off-tumor toxicity
against normal cells 15. The low toxicity, with clinical activity, observed in this patient is most
probably related to multiple factors: (i) CYAD-01 cells demonstrate only short-term
persistence and low proliferation post-infusion, (ii) the chimeric CYAD-01 construct consists
entirely of human sequences, with an entirely native extracellular domain not expected to
induce the anaphylaxis reactions observed with other CAR-Ts using scFv from murine origin
16 , and (iii) the absence of previous lymphodepleting chemotherapy.
While reduction of blasts occurred in AML patients treated with LeY-specific CAR-Ts was
also previously reported, the clinical response could not be entirely attributed to the activity of
the CAR-T since these patients were in morphological CR after reinduction with fludarabine
prior to CAR-T administration 17. In the only published r/r AML case report evaluating the
CD33-specific CAR-Ts without conditioning therapy, a 2-weeks transient decrease in blast
count was reported in the context of grade 4 chills, fevers and transient hyperbilirubinemia,
but with a rapid disease progression, suggesting tumor escape mechanism 18. Of clinical
interest in the setting of MLFS and blood counts approaching complete remission at the time
of allo-HSCT, the patient had identification of a IDH2 mutation that was not present prior to
treatment. This suggests that there can be heterogeneity of NKG2D ligand expression that is
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dependent on the underlying molecular architecture. This is supported by a recent study in
IDH mutant gliomas where NKG2D ligands were down-regulated 19. Future study is required
to further investigate molecular drivers of NKG2D ligand expression in AML in order to
further augment efficacy of CYAD-01. In summary, we report the first objective response to
CAR-T in r/r AML using CYAD-01 without preconditioning chemotherapy and with no
significant toxicities, highlighting the potential of targeting NKG2DL in AML.
Conflict of Interest
CL, EB, SA, BV, DEG and FFL are employed by Celyad SA. The THINK clinical trial is
sponsored by Celyad SA.
References
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NKG2D activating receptor. Annu Rev Immunol. 2013;31:413-441.
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Ligand Expression and Release in Leukemia: Implications for NKG2D-Mediated NK
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levels in acute myeloid leukemias. Blood. 2005;105(9):3615-3622.
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6. Lonez C, Verma B, Hendlisz A, et al. Study protocol for THINK: a multinational open-
label phase I study to assess the safety and clinical activity of multiple administrations
of NKR-2 in patients with different metastatic tumour types. BMJ Open.
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7. Sentman CL, Meehan KR. NKG2D CARs as cell therapy for cancer. Cancer J.
2014;20(2):156-159.
8. Le Bert N, Gasser S. Advances in NKG2D ligand recognition and responses by NK
cells. Immunol Cell Biol. 2014;92(3):230-236.
9. Raulet DH. Roles of the NKG2D immunoreceptor and its ligands. Nat Rev Immunol.
2003;3(10):781-790.
10. Barber A, Zhang T, Megli CJ, Wu J, Meehan KR, Sentman CL. Chimeric NKG2D
receptor-expressing T cells as an immunotherapy for multiple myeloma. Exp Hematol.
2008;36(10):1318-1328.
11. Nikiforow S, Murad J, Daley H, et al. A first-in-human phase I trial of NKG2D
chimeric antigen receptor-T cells in AML/MDS and multiple myeloma. J Clin Oncol.
2016;34(15 Suppl):TPS3102.
12. Maude SL, Frey N, Shaw PA, et al. Chimeric antigen receptor T cells for sustained
remissions in leukemia. N Engl J Med. 2014;371(16):1507-1517.
13. Mardiros A, Forman SJ, Budde LE. T cells expressing CD123 chimeric antigen
receptors for treatment of acute myeloid leukemia. Curr Opin Hematol. 2015;22(6):484-
488.
14. Fan M, Li M, Gao L, et al. Chimeric antigen receptors for adoptive T cell therapy in
acute myeloid leukemia. J Hematol Oncol. 2017;10(1):151.
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15. Brudno JN, Kochenderfer JN. Toxicities of chimeric antigen receptor T cells:
recognition and management. Blood. 2016;127(26):3321-3330.
16. Lamers CH, Willemsen R, van Elzakker P, et al. Immune responses to transgene and
retroviral vector in patients treated with ex vivo-engineered T cells. Blood.
2011;117(1):72-82.
17. Ritchie DS, Neeson PJ, Khot A, et al. Persistence and efficacy of second generation
CAR T cell against the LeY antigen in acute myeloid leukemia. Mol Ther.
2013;21(11):2122-2129.
18. Wang QS, Wang Y, Lv HY, et al. Treatment of CD33-directed chimeric antigen
receptor-modified T cells in one patient with relapsed and refractory acute myeloid
leukemia. Mol Ther. 2015;23(1):184-191.
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surveillance by downregulation of NKG2D ligand expression. Neuro Oncol.
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Figure Legends
Figure 1. Relapsed AML patient treated with CYAD-01 CAR-T therapy. CD34 IHC of
the core biopsy shows clusters of blasts (~7%) consistent with relapsed disease (Panel A).
Prominent dysmegakaryopoiesis was also noted (Panel B). BM biopsy at day +56 following 3
infusions of CYAD-01 showed normal trilineage hematopoiesis with CD34 IHC showing no
increase in blasts (Panel C). Patient also with significant improvement of hematopoiesis as
transfusion independent with marked increase of hemoglobin and platelet count (Panel D).
Fig. 2: Treatment Course and immunohistochemistry analysis of NKG2D ligand
expression in bone marrow sample taken before first CYAD-01 injection. Timeline of
history of treatments and responses is shown (A). BM biopsy of patient prior to CYAD-01
treatment was subject to a range of IHC staining protocols specific for the following
NKG2DL: MICA/MICB, ULBP1, ULBP2/5/6, ULBP3. In addition, a sample of each was
prepared applying standard hematoxylin and eosin (H&E) staining (data not shown). All
samples were examined and graded by light microscopy.100 presumptive neoplastic cells
were counted and analysed, in terms of staining characteristics, using a “H-score” approach.
The intensity of membranous staining was graded for each cell, as follows: 0 – no staining; 1
– minimal staining (each positive cell scored “1”); 2 – mild staining (each positive cell scored
“2”); 3 – moderate/marked staining (each positive cell scored “3”). The percentage of positive
cells for each intensity staining and each ligand is represented in (B). Representative staining
of MICA/B is shown in (C).
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Supplementary information for:
NKG2D-based chimeric antigen receptor therapy induced remission in a
relapsed/refractory acute myeloid leukemia patient.
Authors and affiliations
David A. Sallman1*, Jason Brayer1*, Elizabeth M. Sagatys2, Caroline Lonez3, Eytan Breman3,
Sophie Agaugué3, Bikash Verma4, David E. Gilham3, Frédéric F. Lehmann3, Marco L Davila5
1Malignant Hematology and 2Hematopathology and Laboratory Medicine, H. Lee Moffitt
Cancer Center and Research Institute, Tampa, FL, USA
3 Celyad, SA, Mont-Saint-Guibert, Belgium
4 Celyad, SA, New York, NY, USA
5Blood & Marrow Transplantation and Cellular Immunotherapy, H. Lee Moffitt Cancer
Center and Research Institute, Tampa, FL, USA
*These authors contributed equally to this work.
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Supplementary Figures
Fig. S1: Patient’s CYAD-01 phenotype and in vitro functionality. (A) A sample of the
infusion product was analyzed for cell subset composition and memory phenotype. CD3+ viable
singlet cells showed a high CD8 preponderance with both CD4 and CD8 subsets possessing a
majority of CD62Llo phenotype. As shown in the CD8 subset, both CD62Lhi and CD62Llo
populations were CD95+ and CD28lo. Together this suggests the infusion product to be
composed primarily of CD8+ T cells that possess an effector memory phenotype. 88% of the
CD4+ T cells and 94% of the CD8+ T cells were NKG2D+. (B) Patient’s CYAD-01 product
was incubated in the presence or absence of NKG2D blocking antibody (CD314 Ab) (light and
dark grey bars, respectively) with PANC-1 and K562 cancer cells (at a 1:1 ratio). After 24h of
incubation, supernatants were harvested and analyzed for IFN-γ secretion. Dotted line
represents the limit of detection (LOD). Each bar represents the mean and SD of one experiment
conducted at least in duplicate, ****: p < 0.001. (C) Patient’s CYAD-01 cells were cultured at
a 1:1 ratio with PANC-1 cells in the presence or absence of CD314 blocking Ab. After a 20h
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incubation the CYAD-01 cells were washed away and the remaining PANC-1 cells stained with
AlamarBlue to quantify the remaining proliferating PANC-1 cells. Each bar represents one
experiment conducted in triplicate with the corresponding SD, *: p < 0.05.
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Fig. S2: Immunohistochemistry analysis of NKG2D ligand expression in bone marrow
sample taken before first CYAD-01 injection. One BM biopsy was subject to a range of IHC
staining protocols specific for the following NKG2DL: MICA/MICB, ULBP1, ULBP2/5/6,
ULBP3. In addition, a sample of each was prepared applying standard hematoxylin and eosin
(H&E) staining (data not shown). All samples were examined and graded by light microscopy.
Representative stainings of ULBP1 (A), ULBP2/5/6 (B) and ULBP3 (C). It has to be noted that
ULBP2/5/6 are predominantly stained in the nucleus and that ULBP1, ULBP3 and MICA/B
(Fig. 2B) display cytoplasmic staining as well.
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Supplementary Methods 1
Study THINK design 2
The THINK (THerapeutic Immunotherapy with NKR-2) trial is an open-label Phase I study 3
which primarily aims to assess the safety and clinical activity of the CYAD-01 treatment 4
administered three times at 2 weeks intervals between each administration without prior 5
lymphodepleting chemotherapy in patients with refractory or relapsing malignancies, including 6
patients with metastatic or locally advanced colorectal cancer, urothelial carcinoma, triple-7
negative breast cancer, pancreatic cancer, recurrent epithelial ovarian and fallopian tube 8
carcinoma, AML/MDS or MM. The study is split into two segments; a dose escalation segment 9
evaluating three dose-levels (3x108, 1x109 and 3x109 cells/injection) to determine the 10
recommended dose of CYAD-01 cells and an expansion phase to investigate the clinical activity 11
across multiple tumor indications while extending the safety study. 12
Manufacture of cell products 13
CYAD-01 (previously known as NKR-2) refers to the viable cell population obtained after 14
retroviral transduction of autologous T-cells with the NKG2D-based CAR. CYAD-01 will be 15
supplied cryopreserved in bags containing a T-cell dose in accordance with the dose-level 16
which is to be administered. 17
Characterization of the patient CYAD-01 18
CYAD-01 identity (% NKG2D on CD4+, CD4+CD8+ and CD8+ T cells), purity (% viable CD3+) 19
and viability (with 7AAD dye) are assessed by flow cytometry. Cell yield is assessed by cell 20
counting (exclusing Trypan blue). In vitro product functionality/potency is evaluated by 21
assessment of IFN-γ secretion via ELISA and metabolic activity of tumor cells via Alamar Blue 22
assay upon co-culture of CYAD-01 with NKG2D ligand-expressing tumor cells (Panc-1 cells 23
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and /or K562). NKR-2 microbiological safety was confirmed by absence of microbiological 1
growth, assessed by BactAlert, absence of mycoplasms, assessed by qPCR based MycoTool 2
assay, and compliant endotoxin level (<8.67 EU/ml), assessed by PTS EndoSafe LAL assay. 3
Patient CYAD-01 met the product specifications. CYAD-01 phenotype identity (90% 4
NKG2D+), purity (95% CD3+), viability (86%) and cell yield (300 x 106 cells), evidenced a 5
viable, highly pure CD3+, NKG2D+ product. Viral vector safety was evaluated by Vector copy 6
number (VCN) and Replication copy number (RCR) qPCR-based assays.In vitro product 7
functionality/potency was confirmed by IFN-γ secretion and efficient cytotoxicity effect in 8
response to NKG2DL on tumor cells. 9
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