Regular Article
IMMUNOBIOLOGY AND IMMUNOTHERAPY
CD70-specific CAR T cells have potent activity against
acute myeloid leukemia without HSC toxicity
Tim Sauer,1,2 Kathan Parikh,1 Sandhya Sharma,1 Bilal Omer,1 David Sedloev,2 Qian Chen,2 Linus Angenendt,3 Christoph Schliemann,3
Michael Schmitt,2 Carsten Müller-Tidow,2 Stephen Gottschalk,4 and Cliona M. Rooney1
1
Center for Cell and Gene Therapy, Baylor College of Medicine, Houston Methodist Hospital–Texas Children’s Hospital, Houston, TX; 2Department of Internal
Medicine V, Heidelberg University Hospital, Heidelberg, Germany; 3Department of Internal Medicine A, University Hospital of Muenster, Muenster, Germany; and
4
Department of Bone Marrow Transplantation and Cellular Therapy, St Jude Children’s Research Hospital, Memphis, TN
The prognosis of patients with acute myeloid leukemia (AML) remains dismal, highlighting
the need for novel innovative treatment strategies. The application of chimeric antigen
l Expression level,
receptor (CAR) T-cell therapy to patients with AML has been limited, in particular by the lack
viability, and
of a tumor-specific target antigen. CD70 is a promising antigen to target AML, as it is
functional capacity
of CD70scFv-CAR
expressed on most leukemic blasts, whereas little or no expression is detectable in normal
constructs are
bone marrow samples. To target CD70 on AML cells, we generated a panel of CD70-CAR
strongly influenced
T cells that contained a common single-chain variable fragment (scFv) for antigen detection,
by their structural
composition.
but differed in size and flexibility of the extracellular spacer and in the transmembrane and
the costimulatory domains. These CD70scFv CAR T cells were compared with a CAR conl CD27z-CAR T cells can
struct that contained human CD27, the ligand of CD70 fused to the CD3z chain (CD27z). The
effectively eliminate
AML cells in vitro and
structural composition of the CAR strongly influenced expression levels, viability, expansion,
in vivo, but spare
and cytotoxic capacities of CD70scFv-based CAR T cells, but CD27z-CAR T cells demonnormal hematopoiesis.
strated superior proliferation and antitumor activity in vitro and in vivo, compared with all
CD70scFv-CAR T cells. Although CD70-CAR T cells recognized activated virus-specific T cells
(VSTs) that expressed CD70, they did not prevent colony formation by normal hematopoietic stem cells. Thus, CD70targeted immunotherapy is a promising new treatment strategy for patients with CD70-positive AML that does not affect
normal hematopoiesis but will require monitoring of virus-specific T-cell responses.
KEY POINTS
Introduction
Treatment of patients with acute myeloid leukemia (AML), the
most common acute leukemia among adults, remains challenging. Changes in standard treatment have been moderate
during the past 3 decades, and the outcome remains poor for
most patients, in particular for those aged .60.1,2 Disease remission can be achieved in .80% of patients with AML with
intensive chemotherapy regimens. However, more than 50% of
patients eventually relapse. This high rate of treatment failure
has been attributed in part to leukemic stem cells (LSCs), a minor
fraction of leukemic cells that maintain and reinitiate the disease.
LSCs are resistant to conventional chemotherapy because of
their quiescent cellular state,3,4 but their elimination is paramount to maintaining long-term remissions.
The adoptive transfer of chimeric antigen receptor (CAR)modified T cells has evolved as a promising treatment of patients
with CD19-expressing malignancies, such as acute lymphoblastic leukemia and B-cell lymphoma. So far, patients with AML
have not benefitted from this innovative treatment strategy. A
major obstacle is the identification of target antigens that are
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29 JULY 2021 | VOLUME 138, NUMBER 4
expressed on the surface of AML bulk cells and LSCs but are
absent or expressed at low levels on normal hematopoietic stem
cells (HSCs) and other healthy tissues. CAR T cells specific for
Lewis Y,5 CD33,6,7 CD123,7 CD44-v6,8 CLL-1,9 FLT-3,10 and folate receptor-b11 have proved to be effective in preclinical AML
models, and CD33 and CD123 are currently being evaluated in
clinical trials as targets for CAR T-cell therapy for AML.12 Despite
their potency in eliminating AML bulk cells and LSCs, CD33- and
CD123-CAR T cells can be used only as a bridge to transplant
therapy, because both target antigens are also expressed on
normal HSCs.
CD70 is a type 2 transmembrane glycoprotein and a member of
the tumor necrosis factor (TNF) ligand family. It is a promising
target for CAR T-cell therapy for AML, because it is expressed on
both leukemic blasts and LSCs in patients with AML.13,14 In
contrast to CD33 and CD123, CD70 is not detectable on normal
HSCs,13,14 suggesting that CD70-specific CAR T cells could
provide a therapy for AML without adversely affecting hematopoiesis. A promising antitumor activity of a CD70-specific
monoclonal antibody, in combination with hypomethylating
© 2021 by The American Society of Hematology
agents for the treatment of patients with AML, has been
reported.15
In the present study, we evaluated CD70 as a novel therapeutic
target for CAR T-cell therapy for AML. We designed a panel of
CD70-CAR T cells containing different antigen binding, extracellular spacer, and intracellular signaling domains. Two of the
CAR constructs exhibited significant antitumor efficacy in vitro
and in vivo, with major effects imposed by antigen binding,
hinge and costimulatory domains. Although AML cells were
effectively eliminated, CD70-CAR T cells spared normal HSCs,
thereby avoiding potentially dangerous on-target/off-tumor
toxicity. Our data support CD70 as a promising target for immunotherapy, as well as early phase clinical testing of CD70-CAR
T cells in patients with CD70-positive AML.
Material and methods
CD70-CAR construction
To generate a panel of CD70scFv CAR constructs, cDNA containing the VH and VL chains derived from the single-chain variable
regions (scFvs) of a published antibody sequence16 was commercially synthesized (IDT, Coralville, IA) and fused to different
spacers (intermediate [IM] and long-flexible [LF]), transmembrane
domains (CD28 and CD27 derived), 1 of 3 costimulatory domains
(CD27, CD28, and 4-1BB), and the CD3z chain, using In-Fusion
Cloning (Takara, Mountain View, CA). The IM spacer contained an
IgG1-derived CH3 and an IgG4 hinge domain, and the LF spacer
consisted of a modified CH2 and CH3 domain17 that are connected through 2 IgG4 hinge domains for additional flexibility.
CAR constructs that included the CD28 and the 4-1BB costimulatory domains contained the CD28 transmembrane domain,
and the transmembrane domain of CAR T cells with the CD27
costimulatory domain was also derived from the CD27 molecule
(supplemental Figure 2A-B, available on the Blood Web site). For
the CD70ligand-CAR (CD27z-CAR), we followed our previously
published design18 and fused the cDNA of the human CD27
protein to CD3z (supplemental Figure 2C). Using the XhoI and NotI
sites, we cloned the fragments into a linearized SFG vector that
contained an internal ribosome entry site sequence and a truncated NGFR or CD19 downstream of the CAR insertion site for
plasmid detection (supplemental Figure 2D).
Retroviral vector production and
T-cell transduction
Retroviral vector production and T-cell transduction were performed as previously described.19,20 In brief, 293T cells were
transfected with packaging plasmids (PeqPam, RD114) and the
SFG vector containing the CAR construct, using GeneJuice (Merck
Millipore, Billerica, MA). The viral supernatant was harvested after
48 hours. Peripheral blood mononuclear cells were isolated from
the peripheral blood by density gradient centrifugation, and
the T cells were activated by CD3 and CD28 antibodies. The
T cells were transduced in 24-well plates coated with RetroNectin
(Takara) after 48 hours of expansion in complete medium (45%
RPMI-1640; Hyclone, Logan, UT), 45% Click’s medium (FujiFilm;
Irvine Scientific, Santa Ana, CA), 2 mM L-glutamine (Invitrogen,
Carlsbad, CA), and 10% fetal bovine serum (Hyclone) supplemented with 10 ng/mL interleukin-7 (IL-7) and 5 ng/mL IL-15
(Peprotech, Rocky Hill, NJ).21
CD70-CAR T CELLS FOR AML
Cell lines
We obtained 293T cells and the AML cell lines THP-1 and KG-1a
from ATCC. The AML cell line Molm-13 was purchased from
DSMZ. The cells were maintained in Iscove’s modified Dulbecco’s
medium, for Molm-13, KG-1a, and 293T, and RPMI 1640 for
THP-1, both supplemented with 2 mM L-glutamine and 10% or
20% fetal bovine serum, according to the manufacturer’s recommendations, and 1% penicillin-streptomycin in a humidified
atmosphere containing 5% CO2 at 37°C. IMS-M2 was kindly
provided by Margaret Goodell (Baylor College of Medicine,
Houston, TX). All cell lines were mycoplasma free, according to
the Mycoalert Detection Kit (Lonza, Basel, Switzerland).
Flow cytometry
Fluorochrome-conjugated isotype controls and antihuman
CD70, CD45, CD4, CD8, CD3, CD19, CD45RO, CD34, NGFR,
CCR7, LAG3, TIM-3, PD-1, interferon-g (IFN-g), and TNF-⍺ antibodies were purchased from BD Biosciences (San Jose, CA),
Beckman Coulter (Brea, CA), Thermo Fisher Scientific (Waltham,
MA), or Biolegend (San Diego, CA). Biotin-labeled protein L
and fluorochrome-labeled streptavidin for CAR detection were
purchased from Thermo Fisher Scientific and BD Biosciences.
Flow cytometric data were acquired using the FACSCanto II (BD
Biosciences) and analyzed with FlowJo, version 10 (Tree Star,
Ashland, OR).
TMA construction, immunohistochemistry, and
evaluation
A tissue microarray was established using pretherapeutic bone
marrow biopsy specimens from patients diagnosed with AML who
had received intensive treatment in the Department of Internal
Medicine A at the University Hospital of Muenster between 2006
and 2016. Informed consent was obtained from all patients, in
accordance with the Declaration of Helsinki. The local ethics
committee approved the study. Bone marrow specimens were
fixed in formaldehyde, decalcified, and embedded in paraffin.
IHCPlus Polyclonal CD27L/CD70 (clone LS-A8809; LSBio, Seattle,
WA) was used as the primary antibody. Staining was performed
with the UltraVision LP Detection System (Thermo Fisher Scientific). Two experienced hematologists without knowledge of the
patients’ clinical data evaluated all tissue microarray samples. The
staining of all bone marrow samples was evaluated using an
H-score.22
Chromium-51 release assay
Short-term cytotoxicity was determined with the chromium-51
release assay. In brief, target cells were incubated with 51Cr
sodium chromate for 1 hour and plated in 96-well plates. CAR or
nontransduced (NT) T cells were added at multiple effector-totarget (E:T) ratios. After 5 hours of incubation, the supernatant
was harvested, and chromium release was detected with a gamma
counter. The mean percentage of specific lysis of triplicate wells
was calculated as previously reported.23
Generation of multivirus-specific T cells
Generation of multivirus-specific T cells (MVSTs) for use in coculture assays was performed as previously described.24,25
Coculture assay
A schematic of the experimental design is shown in supplemental
Figure 5. In brief, CAR or NT T cells were cocultured with tumor
cells in 96-well plates in the absence of exogenous cytokines. We
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29 JULY 2021 | VOLUME 138, NUMBER 4 319
harvested 1 well per condition every 5 days and calculated the
total number of T cells and tumor cells by flow cytometry with
CountBright beads (Invitrogen). The dead cell population was
excluded by 7AAD-staining. Tumor cells were identified by
ZsGreen expression, and T cells were detected with an CD3
antibody. If tumor cells were eliminated, T cells were rechallenged
with the same number of fresh tumor cells that was initially used.
For coculture with MVSTs, we labeled the MVSTs with CellTrace
Violet (Thermo Fisher Scientific) before setup of the coculture, to
distinguish CAR or NT T cells from MVSTs. One well per condition
was harvested after 3 days; the remaining MVSTs were stimulated
with Epstein-Barr virus (EBV), adenovirus, and cytomegalovirus
(CMV) pepmixes overnight, and intracellular cytokine staining for
IFN-g and TNF-⍺ was performed by flow cytometry.
All procedures complied with the requirements of the Institutional Animal Care and Usage Committee of Baylor College of Medicine.
CRISPR/Cas9-mediated T-cell receptor knockout
Results
CRIPSR/Cas9-mediated gene editing on T cells has been extensively described elsewhere.26 In brief, CD27z-CAR T cellsTs
and NT T cells were electroporated with precomplexed TrueCut
Cas9 protein (Thermo Fisher Scientific) and guide RNA, which
targets the ⍺-chain of the human T-cell receptor (TCR).27 After
electroporation and verification of successful TCR knockout,
fluorescence-activated cell sorting was used to obtain pure TCRnegative and TCR-positive CAR T-cell populations.
Expression of CD70 on AML cell lines and AML
bone marrow samples
Cytokine release and enzyme-linked
immunosorbent assays
Supernatants were harvested at selected time points after the
first and second cocultures and analyzed for multiple cytokines
and chemokines (GM-CSF, IFN-g, TNF-⍺ and IL-1b, -2, 4, -5, -6,
-7, -8 -10, -12 [p70], and -13), using the Multiplex Biomarker
Immunoassay System (Merck Millipore, Billerica, MA), according
to the manufacturer’s recommendation. Levels of soluble CD27
(sCD27) were determined with the Human CD27/TNFRSF7
DuoSet ELISA Kit (R&D Systems, Minneapolis, MN), according to
the manufacturer’s recommendation.
Colony formation assay with normal HSCs
Normal HSCs were selected from the cord blood of healthy individuals by using CD34-specific microbeads (Miltenyi, BergischGladbach, Germany). After separation, CD34-positive HSCs were
incubated with CAR or NT T cells, or medium only in CTL medium
at an E:T ratio of 10:1 for 6 hours. After incubation, cell suspensions were plated in duplicate in MethoCult H4434 medium
supplemented with recombinant cytokines (Stemcell Technologies, Vancouver, BC, Canada). After 14 days of incubation, the
median total number of colonies per condition was counted using
an inverted microscope. After they were counted, the cells were
harvested and seeded for the second plating.
Xenograft model of AML and
bioluminescent imaging
NOD.Cg-Prkdcscid IL2rgtm1Wjl/SzJ (NSG) mice (6-10 weeks of age)
were purchased from The Jackson Laboratory (Bar Harbor, ME)
and maintained at the Baylor College of Medicine Animal Facility. If required for tumor establishment, animals were sublethally irradiated (200 cGy). Animals received injection of
tumor and T cells via the tail vein, as described in “Results.”
Leukemia burden or T-cell expansion and trafficking were
monitored by bioluminescent imaging (BLI; photons per
second per cm2 spectral radiance), using the Xenogen In Vivo
Imaging System (IVIS; Caliper Life Sciences, Hopkinton, MA).
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Statistical analysis
Prism 5 (GraphPad Software, San Diego, CA) was used for statistical analysis. For comparisons between 2 groups, we used the
2-tailed Student t test. For 3 or more groups, 2-way analysis of
variance (ANOVA) with Tukey’s multiple-comparison test was
used. For animal experiments, we analyzed survival from the time
of T-cell injection by constructing Kaplan-Meier curves and using
log-rank (Mantel-Cox) tests.
To validate CD70 as a target antigen for CAR T-cell therapy for
AML, we evaluated CD70 expression on AML cell lines, bone
marrow samples obtained from patients with AML, and normal
bone marrow samples from patients without AML. CD70 expression was detectable in 3 of 4 AML cell lines that we tested
(Molm-13, THP-1, and IMS-M2), whereas KG-1a was negative
(Figure 1A). CD70 expression was significantly higher in primary
bone marrow samples from 136 patients with newly diagnosed
AML than in normal bone marrow (n 5 8) in which CD70 was not
detected (Figure 1B-C; P , .0001). By flow cytometry, CD70
expression varied in primary AML samples, both in the frequency
of CD70-positive cells and expression level (Figure 1D; see
supplemental Figure 1 for the gating strategy).
Generation and characterization of
CD70-CAR T cells
We generated a panel of 6 CD70scFv-based CAR constructs with
different spacers (IM and LF) and signaling domains (CD27, CD28,
or 4-1BB) (IM-28z, IM-BBz, IM-27z, LF-28z, LF-BBz, and LF-27z;
supplemental Figure 2A-B). In addition, we generated 1 CAR that
consisted of CD27, the CD70 receptor, linked to the CD3z signaling domain (CD27z; supplemental Figure 2C). CAR T cells were
generated by retroviral transduction, with the median percentage
of CAR-positive T cells ranging between 44.4% and 95.2% (Figure
2A-B; supplemental Figure 3A). In downstream experiments, we
adjusted the number of T cells according to the percentage of
CAR-positive cells. All CAR T-cell populations, except for LF-27zand CD27z-CAR T cells, had a significantly lower viability than
the NT T cells (Figure 2C); in particular, the IM spacer and the
4-1BB costimulatory domain contributed to decreased viability
(Figure 2C), which translated into reduced T-cell expansion after
transduction (Figure 2D). Even though CD70 expression is known
to be upregulated on activated T cells, CD70-CAR T cells in
contrast to NT T cells expressed minimal levels of CD70 (supplemental Figure 3B). Further phenotypic analysis revealed that all
CD70scFv-based CAR T cells, except for LF-27z, induced T-cell
differentiation, as judged by an increased frequency of effector
memory–like (CD45RO1, CCR72) and terminally differentiated–
like (CD45RO2 CCR72) T cells in comparison with NT T cells
and CD27z-CAR T cells (Figure 2E; supplemental Figure 3C).
CD4/CD8 composition was not significantly different between
T cells expressing the different CAR constructs (supplemental
Figure 4A-B).
SAUER et al
A
Molm-13
Count
100
THP-1
IMS-M2
KG-1a
100
100
100
80
80
80
80
60
60
60
60
40
40
40
40
20
20
20
20
Isotype
CD70
0
0
-10
1
0 103
104
105
0
1
-10
0 103
104
105
0
3
0
10
104
105
1
-10
0 103
104
105
CD70 expression
C
B
AML
Normal bone marrow
300
****
CD70 expression (H-score)
20x
20x
200
100
0
normal BM
(n=8)
63x
AML
(n=136)
D
Pat. 0001
Pat. 0003
100
Isotype
CD70
80
60
Count
40
91.6%
20
Pat. 0012
100
100
80
80
60
60
40
35.6%
40
20
MFI 457
20
23.0%
MFI 1000
0
0
0
10
3
104
105
MFI 271
0
0
10
3
104
105
0
10
3
104
105
CD70 expression
Figure 1. CD70 expression of AML cell lines and primary bone marrow samples from patients with AML. (A) Evaluation of CD70 expression in several primary AML cell lines
by flow cytometry. (B) Representative images of immunohistochemical staining for CD70 expression of bone marrow samples derived from patients without AML (normal bone
marrow) and patients with newly diagnosed AML. Leukemic blasts in patients with AML were identified by their morphology. (C) Comparison of CD70 expression in bone marrow
samples of 136 patients with AML and 8 individuals with normal bone marrow findings by immunohistochemical staining. The H-score was used to determine the staining
intensity. ****P , .0001, by unpaired Student t test. (D) Analysis of CD70 expression in bone marrow samples of patients with newly diagnosed AML by flow cytometry.
Percentage of CD70-expressing cells and median fluorescence intensity (MFI) of the CD70-positive cell population are depicted.
LF-BBz- and CD27z-CAR T cells have superior
antitumor activity in vitro
To investigate the cytolytic capacity of our CD70-CAR T cells, we
used a standardized chromium-51 release assay.28 All CAR T-cell
populations mediated cytotoxicity against the CD70-positive
tumor cell lines (Figure 2F; Molm-13, THP-1) but not against
CD70-negative cells (Figure 2G; KG-1a). T cells expressing IM
spacer–based CAR T cells with the CD28 costimulatory domain
had a significantly greater cytolytic activity than all other CAR
constructs. Because CAR T-cell efficacy is determined not only
by its cytolytic activity, but also by the capacity to proliferate
upon tumor challenge, we performed serial coculture assays, in
CD70-CAR T CELLS FOR AML
which CD70-CAR T cells were repeatedly challenged with Molm13 cells. Cocultures were terminated when CAR T cells failed to
eliminate tumor cells or to persist after tumor cell elimination
(supplemental Figure 5). T cells expressing all of the CAR
constructs eliminated tumor cells and proliferated during the first
coculture, but differentially lost their capacity to kill and proliferate during subsequent cocultures. LF-BBz- and the CD27zCAR T cells eliminated tumor cells for 5 consecutive cocultures in
at least 2 of the 4 donors in contrast to other CAR T-cell populations for which tumor outgrowth occurred invariably with the
third coculture (Figure 3A). Increased antitumor activity of
LF-BBz- and CD27z-CAR T cells was mirrored by an increased
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29 JULY 2021 | VOLUME 138, NUMBER 4 321
A
IM-28z
100
CAR–
11.7
IM-BBz
100
CAR+
88.3
CAR–
10.9
IM-27z
100
CAR+
89.1
CAR–
27.1
LF-28z
100
CAR+
72.9
CAR–
17.5
LF-BBz
CAR+
82.5
100
CAR–
9.59
LF-27z
CAR+
90.4
100
CAR–
54.4
CD27z
100
CAR+
45.6
80
80
80
80
80
80
80
60
60
60
60
60
60
60
40
40
40
40
40
40
40
20
20
20
20
20
20
20
0
0
0
0
0
0
0
103
104
0
105
103
104
0
105
103
104
0
105
103
104
0
105
103
104
CAR+
70.3
0
0
105
CAR–
29.7
103
104
105
0
103
104
105
CAR expression
C
80
80
% of T cells
100
% of T cells
100
60
40
20
** *** **
*
*
ns ns
6000
40
20
N
T
IM C
-2
IM 8z
-B
IM Bz
-2
7
LF z
-2
LF 8z
-B
B
LF z
-2
CD 7z
27
z
-2
7
CD z
27
z
LF
8z
LF
-B
Bz
7z
LF
-2
Bz
-2
IM
-2
IM
IM
-B
8z
0
Donor 2
NTC
IM-28z
IM-BBz
IM-27z
LF-28z
LF-BBz
LF-27z
CD27z
8000
60
0
Donor 1
D
Fold change
B
Donor 3
Donor 4
4000
2000
Donor 5
0
d2
E
Naive/CM T cells
***
*
** ***
*
ns
ns
80
60
40
20
*
** ***
*
ns
d7 d15 d22
Days post activation
ns
80
60
40
20
0
40
60
40
NTC
****
****
****
****
****
****
****
IM-28z IM-BBz IM-27z LF-28z LF-BBz LF-27z CD27z
***
****
*
****
****
****
ns
ns
ns
ns
ns
***
ns
**
ns
**
****
**
5:
1
5:
1
E:T ratio
E:T ratio
p-val.
NTC
IM-28z
IM-BBz
IM-27z
LF-28z
LF-BBz
LF-27z
CD27z
2.
5:
5:
2.
10
20
40
40
:1
20
:1
10
:1
0
1
0
1
0
:1
20
:1
20
:1
20
ns
ns
ns
p-val.
NTC
IM-28z
IM-BBz
IM-27z
LF-28z
LF-BBz
LF-27z
CD27z
NTC
****
****
****
****
****
****
****
5:
1
60
5:
1
40
NTC
IM-28z
IM-BBz
IM-27z
LF-28z
LF-BBz
LF-27z
CD27z
80
2.
80
KG-1a (CD70-)
100
Lysis (%)
80
Lysis (%)
100
60
G
THP-1 (CD70+)
Molm-13 (CD70+)
100
40
:1
20
:1
10
:1
F
N
T
IM C
-2
IM 8z
-B
B
IM z
-2
7
LF z
-2
LF 8z
-B
B
LF z
-2
CD 7z
27
z
N
T
IM C
-2
IM 8z
-B
IM Bz
-2
7
LF z
-2
LF 8z
-B
B
LF z
-2
CD 7z
27
z
0
Lysis (%)
***
100
% of CAR T cells
% of CAR T cells
100
EM/EMRA T cells
E:T ratio
IM-28z IM-BBz IM-27z LF-28z LF-BBz LF-27z CD27z
****
****
****
****
****
****
ns
ns
ns
***
****
****
**
****
****
n.s.
****
****
****
***
ns
ns
Figure 2. Generation and characterization of CD70-CAR T cells. (A) Representative histograms of CD70-CAR expression levels after transduction of activated T cells.
CD70scFv-CAR T cells were detected by staining with biotin-labeled protein L, followed by staining with APC-conjugated streptavidin. CD27z-CAR expression was confirmed by
detection of a truncated CD19 using an APC-conjugated CD19 specific antibody. (B) The frequency of CD70-CAR expressing T cells of 4 different donors. (C) The frequency of
viable cells for CD70-CAR and NT T cells of 4 different donors, as determined by forward vs side scatter gating. (D) Fold change in the number of CD70-CAR T cells in culture
during the manufacturing process without antigen stimulation. CAR T cells from 4 different donors were counted in weekly intervals. (E) Phenotypical characterization of CD70CAR T cells by flow cytometry. The frequency of CD70-CAR T cells with a less (naive/CM T cells; left) and a more (effector memory/terminally differentiated effector memory cells
reexpressing CD45RA [EM/EMRA] T cells; right) differentiated phenotype, as determined by the expression of CD45RO and CCR7 in 4 different donors. The colored bars
represent the mean of results from 4 different donors, and the error bars indicate the standard deviation (SD). *P , .05; **P , .01; ***P , .001; ****P , .0001; ns, not significant, by
unpaired Student t test. (F-G) A chromium-51 release assay was used to determine antigen-dependent lysis of CD70-CAR T cells against CD70-positive AML cell lines (Molm-13,
THP-1) (F) and KG-1a (CD702) cells (G) as the control. The graph shows the mean results of 3 technical replicates from 4 different donors, and the error bars indicate the SD.
P values were calculated by 2-way ANOVA and are shown in the table below the graphs. *P , .05; **P , .01; ***P , .001; ****P , .0001; ns, not significant.
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SAUER et al
ability to expand after tumor cell challenge (Figure 3B). As it has
been reported that T-cell activation leads to the release of
soluble CD27 (sCD27) mainly by proteolytic events,29 we sought
to determine whether this process interferes with the functionality of the CD27z-CAR. Membrane-bound CD27 (mCD27) expression before antigen stimulation was significantly higher in
CD70-CAR T cells than in NT T cells. As expected, due to detection of endogenous and CAR-associated CD27, the highest
levels of mCD27 were detectable in CD27z-CAR T cells, which
also resulted in a significantly higher secretion of sCD27 (supplemental Figure 6A-B). Expression of mCD27 on and secretion of
sCD27 by CD27z-CAR T cells remained at high levels at different
time points after antigen stimulation, suggesting that CD27z-CAR
T cells are not functionally impaired by interaction with the CD70
antigen (supplemental Figure 6C-D).
We also measured cytokine production by CD70-CAR T cells
after the first 2 stimulations. CD27z- and LF-27z-CAR T cells
produced the highest levels of TH1/TC1 cytokines, such as IFN-g
and TNF-a, after both stimulations (Figure 3C-D; supplemental
Figures 7 and 8). To further investigate the functional differences
between T cells expressing the different CD70-CAR constructs in
the coculture assays, we examined the expression of exhaustion
markers LAG-3, TIM-3, and PD-1, 5 days after each of the first
3 stimulations. At the end of cocultures 1 and 2, LAG-3, TIM-3,
and PD-1 expression patterns did not differ significantly among
the CAR T-cell populations (supplemental Figure 9A). However,
after the third stimulation, the frequency of triple-negative cells
(LAG-32/TIM-32/PD-12) in the CD27z-CAR T-cell product was
significantly higher, and the percentage of triple-positive cells
was lower than that of the other CD70-CAR T cells (Figure 3E-F).
CD4/CD8 composition did not differ between the CAR constructs after antigen stimulation (supplemental Figure 9B). Even
though target and effector cells were not HLA matched, alloreactivity did not contribute to cytotoxicity of CD70-CAR T cells,
as CD27z-CAR T cells, with and without CRISPR/Cas9-mediated
disruption of the endogenous T-cell receptor, exhibited similar
antitumor efficacy and T-cell expansion capacity (supplemental
Figure 10).
click beetle green luciferase and ZsGreen. Five days after tumor
cell injection, the animals received LF-28z-, CD27z-, or NT T cells
(Figure 4E). In contrast to NT T cells, LF-28z- and CD27z-CAR
T cells rapidly eliminated THP-1 cells (Figure 4F). After having
established the most potent antitumor efficacy of LF-28z- and
CD27z-CAR T cells against AML cell lines, we sought to determine
their cytotoxic capacity against patient-derived AML blasts. We
challenged CAR T cells with blasts expressing different levels of
CD70 (Figure 1D) and found that, in contrast to NT T cells, LF-28zand CD27z-CAR T cells eliminated leukemic blasts from patients
with AML, even with low CD70 expression (Figure 4G).
CD27z-CAR T cells induce significant T-cell
expansion in vivo
To determine whether the improved antitumor activity of LF-28zand CD27z-CAR T cells is associated with greater T-cell expansion and persistence, we used our Molm-13 xenograft model
with unmodified tumor cells and CD70-CAR T cells that were
genetically modified to express a GFP firefly luciferase fusion
protein (Figure 5A; supplemental Figure 11A-B). Expansion
of CD27z-CAR T cells was significantly greater than that of
CD70scFv-based CAR and NT T cells (Figure 5B-C; P , .0001),
and infusions were well tolerated, given that none of the mice
had significant weight loss (Figure 5D). On day 21 after T-cell
infusion, we measured the frequency of human T cells and tumor
cells in the peripheral blood of mice infused with LF-28z-, LF-BBz-,
and CD27z-CAR T cells. More than 95% of circulating human cells
in mice infused with CD27z- and LF-28z-CAR T cells were human
T cells; in contrast, most of the human cells in mice infused with
LF-BBz-CAR T cells were AML blasts (supplemental Figure 11C-D).
CD70-CAR T cells recognize activated virus-specific
T cells, but do not recognize normal HSCs
To evaluate antileukemic activity of our CD70-CAR T cells in vivo,
we used a murine xenograft model, in which NSG mice received
IV injections of Molm-13 cells that were genetically modified to
express click beetle green luciferase and ZsGreen. On day 4,
animals received a single IV dose of 5 3 106 CD70-CAR or NT
T cells. Tumor growth was observed by weekly BLI (Figure 4A).
Only LF-28z- and the CD27z-CAR T cells efficiently controlled
leukemic growth, leading to complete leukemia remission in all
mice by day 21 (Figure 4B-C), resulting in a significant survival
advantage compared with mice treated with NT or other CD70CAR T cells (Figure 4D). In contrast, IM-28z-, LF-BBz-, and LF-27zCAR T cells had only transient antitumor activity. In vivo testing of
IM-BBz- and IM-27z-CAR T cells could not be performed because of limited in vitro expansion after CAR transduction
(Figure 2D).
CD70 expression on activated T cells has been reported,30 and,
as expected, we observed decreased viability and poor expansion of several of our CD70-CAR T-cell populations. To
determine whether CD70-CAR T cells kill activated T cells, we
focused on MVSTs that recognize EBV, adenovirus, and CMV.
We first measured CD70 expression by MVSTs after activation by
incubating peripheral blood mononuclear cells with EBV, adenovirus, and CMV pepmixes. By 4 days after stimulation, CD70
expression was detectable on a median of 38.9% (range, 27.1%
to 41.0%) of T cells (Figure 6A-B). Expression increased (percentage and intensity) over time and by day 17 after first
stimulation, 97.4% (range, 89.3% to 98.3%) of cells were CD70positive (Figure 6A-B). To investigate whether CD70-CAR T cells
kill MVSTs, we cocultured IM-28z-, LF-28z-, and CD27z-CAR
T cells with autologous, CellTrace Violet–labeled MVSTs for
3 days and evaluated their survival by flow cytometry. Compared
with NT T cells, all CD70-CAR T-cell populations reduced the
number of MVSTs significantly (Figure 6C-D). To further investigate this finding, we determined the ability of MVSTs to
produce IFN-g and TNF-⍺ in response to viral pepmixes after
coculture with CD70-CAR T cells. CD70-specific CAR T cells did
not lead to a significant reduction of responsive MVSTs in comparison with NT T cells (Figure 6E), suggesting that CD70-CAR
T cells may impair but not completely abrogate viral immunity.
We used a second xenograft model to confirm the potent antitumor activity of LF-28z- and CD27z-CAR T cells in vivo. After
sublethal irradiation with 2 Gy, NSG mice received an injection of
THP-1 cells, which had been genetically modified to express
In contrast to CD33 and CD123, which are frequently used as
target antigens for AML, CD70 expression is not detectable on
normal human HSCs.13 Thus, to validate the safety of our CD70CAR T cells, we examined their cytotoxicity towards normal
CD27z- and LF-28z-CAR T cells have potent
antitumor efficacy in vivo and against primary AML
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29 JULY 2021 | VOLUME 138, NUMBER 4 323
A
Tumor cells
(fold expansion)
40
NTC IM-28z
IM-BBz
IM-27z
LF-28z
20
10
1
123
12345
1234
123
12345
1234
12345
Donor 1
# of co-culture
Donor 2
Donor 3
B
Donor 4
50
Donor 5
NTC IM-28z
IM-BBz
IM-27z
LF-28z
LF-27z
LF-BBz
CD27z
40
30
20
10
0
1
123
12345
1234
123
12345
1234
12345
# of co-culture
D
**
6×10
**** ****
4×104
2×104
**** ****
**** ****
60
40
20
0
HSCs. For this purpose, CD34-positive HSCs, isolated from cord
blood units (CBU; n 5 2), were cultured with T cells expressing
LF-28z- or CD27z-CAR T cells, the 2 CAR T cells with the most
promising antitumor activity. A third-generation, CD33-directed
CAR (CD33CART; M.S., unpublished data), which is known to
cause toxicity in normal hematopoiesis, NT T cells, and T-cell
culture medium served as the control. After 6 hours of incubation, a serial colony-forming unit (CFU) assay was performed
to determine the deleterious effect of CD70- and CD33-CAR
T cells on human hematopoietic stem and progenitor cell selfrenewal potential in vitro. With respect to the number of colonies
counted 14 days after primary plating, we did not observe
significant differences between NT and CD70-CAR T cells,
whereas CD33-CAR T cells led to a significant reduction in the
number of colonies (Figure 6F; P , .0001; supplemental
Figure 12A), suggesting that CD70-CAR T cells do not impair
normal hematopoiesis. The count after the second plating
29 JULY 2021 | VOLUME 138, NUMBER 4
**
6×104
***
4
4×10
2×104
****
**
****
IFNγ
IL2
GM-CSF
TNFα
TH1/TC1
IL4
IL5
IL6
IL10
IL13
TH2/TC2
F
% LAG3+/TIM3+/PD-1+
80
IM
IM 28z
IM BBz
LF 27z
LF 28z
-B
LF Bz
CD27z
27
z
% LAG3-/TIM3-/PD-1-
100
***
**
***
**
**
***
***
8×104
IM
-2
IM 8z
-B
B
IM z
-2
7
LF z
-2
8
LF z
-B
Bz
LF
-2
7
CD z
27
z
4
60
50
40
30
20
10
0
ns
Donor 3
Donor 4
Donor 6
IM
IM 28z
-B
IM Bz
-2
LF 7z
LF 28z
-B
LF Bz
CD27z
27
z
8×104
2nd stim
1×105
N
IM TC
-2
IM 8z
-B
IM Bz
-2
LF 7z
-2
LF 8z
-B
LF Bz
-2
CD 7z
27
z
Cytokine conc (pg/mL)
1×105
Cytokine conc (pg/mL)
C
E
blood®
CD27z
30
1st stim
324
LF-27z
LF-BBz
0
T cells (fold expansion)
Figure 3. Functional characterization of CD70-CAR
T cells after repeated antigen stimulation in vitro.
CD70-CAR/NT T cells and AML cells (Molm-13) were
cocultured for 5 days, and fresh Molm-13 cells were
repeatedly added every 5 days thereafter. The absolute
cell count of tumor and T cells at the end of each
coculture was determined by flow cytometry with
CountBright counting beads. (A) The absolute cell
count of Molm-13 cells at the end of each coculture. (B)
The absolute cell count of CD70-CAR/NT T cells at the
end of each coculture. (C-D) Cytokine production by
CD70-CAR and NT T cells after antigen stimulation was
determined with the multiplex detection assay. Data
show the quantitative determination of TH1/TC1– and
TH2/TC2–associated cytokines 24 hours after the first (C)
and second (D) antigen stimulation. Data are the mean
of results from 4 different donors. P values indicating the
statistical significance for secreted cytokines of CD27zCAR-T cells in comparison with all other constructs and
NT T cells were calculated by 2-way ANOVA. (E-F)
Expression of LAG-3, TIM-3 and PD-1, markers associated with T-cell exhaustion on CD70-CAR T cells after
antigen stimulation. Data are the mean frequency 6
standard deviation (SD) of triple-negative (E) and triplepositive (F) T cells from 3 different donors 72 hours after
the third antigen stimulation. The error bars indicate the
SD. **P , .01; ***P , .001; ****P , .0001; ns, not significant, by unpaired Student t test.
revealed a lower number of colonies only in the CD27z-CAR
condition for CBU donor 1, whereas no significant differences
were detectable in CBU donor 2 and any of the other conditions
(supplemental Figure 12B-C).
Discussion
In this study, we designed a panel of CD70-CAR T cells and
evaluated the anti-AML activity of T cells expressing these CAR
T cells in vitro and in vivo. We demonstrated that CAR design
influences CD70-CAR T-cell function and that a CAR based on the
CD70 receptor CD27 has superior antitumor activity compared
with standard, scFv-based CAR T cells.
The identification of optimal target antigens for CAR T-cell
therapy for AML has been challenging, as the majority of
targetable surface proteins are expressed not only on
SAUER et al
A
1x106
Molm-13.
zsg.CBG
5x106
T-cells
-4
0
NTC
IM-28z
B
Bioluminescence
Imaging
7
14
21
28
LF-28z
35
42
LF-BBz
50
LF-27z
CD27z
Luminescence
5
5.0x10
d0
5.0x104
Luminescence
5
5.0x10
d7
d7
5.0x104
Luminescence
7
1.0x10
d14
1.0x106
Luminescence
7
1.0x10
d21
1.0x106
Luminescence
7
1.0x10
d50
D
1011
9
10
108
107
106
100
Percent survival
NTC
IM-28z
LF-28z
LF-BBz
LF-27z
CD27z
1010
50
25
0
5
10
0 15 30 45 60 75
Days after tumor cell injection
Days after T cell infusion
F
ROI expansion (p/sec/cm2/sr)
E
6
2 Gy
NTC
IM-28z
LF-BBz
LF-27z
** **
LF-28z
CD27z *
(n=5/group)
75
d0
d7
d1
4
d2
1
d2
8
d3
5
d4
2
d5
0
C
ROI expansion (p/sec/cm2/sr)
1.0x106
1x10
THP-1. 5x106 Bioluminescence
zsg.CBG T-cells
Imaging
-4
0
3
10
17
1010
109
NTC (n=4)
LF-28z (n=5)
CD27z (n=5)
108
107
106
105
d-1
d3
d10 d17
Days after T cell infusion
G
AML pat. 0001
AML pat. 0003
AML pat. 0012
*
40
60
**
Lysis (%)
Lysis (%)
60
80
***
40
60
40
20
0
0
0
LF
-2
N
N
TC
LF
-2
8z
CD
27
z
20
8z
CD
27
z
20
***
**
N
TC
LF
-2
8z
CD
27
z
80
*
TC
Lysis (%)
80
Figure 4. Antileukemic activity of CD70-CAR T cells in 2 murine AML xenograft models and against primary AML blast cells. (A) The experimental setup of the Molm-13
xenograft model. CD70-CAR T cells (5 3 106) were injected 4 days after engraftment of luciferase-labeled Molm-13 cells (1 3 106). BLI was performed before T-cell injection
on day 0 and weekly thereafter (n 5 5 for each treatment group). (B) BLI on the indicated days (after T-cell injection) of mice with Molm-13 cell engraftment and treatment with
CD70-CAR T CELLS FOR AML
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29 JULY 2021 | VOLUME 138, NUMBER 4 325
A
1x106
Molm-13 WT
5x106 luciferase
labeled T-cells
-4
B
Bioluminescence
Imaging
0
NTC
4
LF-28z
11
7
LF-BBz
15
LF-27z
CD27z
d0
d4
5.0x106
d7
d11
d15
5.0x104
D
ROI expansion (fold change)
1000
100
****
10
1
0.1
140
Body weight (percentage)
C
120
NTC
LF-28z
LF-BBz
LF-27z
CD27z
(n=5/group)
100
80
0.01
d0
d4
d7
d11
d15
Days after T cell infusion
d0
d4
d7 d11 d15 d18 d25 d32
Days after T cell infusion
Figure 5. Expansion and trafficking of CD70-CAR T cells in vivo. (A) The experimental setup. NSG mice underwent AML engraftment with 1 3 106 Molm-13 cells and
injection of 5 3 106 CD70-CAR or NT T cells genetically modified to express a luciferase-GFP fusion protein 4 days later. T-cell expansion and trafficking were monitored by
BLI on days 4, 7, 11, and 15 (n 5 5 for each treatment group). (B) BLI of mice on the indicated days after infusion of CD70-CAR or NT T cells. (C) Quantitative analysis of BLI
for each treatment group. Data are the mean 6 standard deviation (SD) of 5 animals per treatment group. The area under the curve was calculated for each treatment
group and compared by unpaired Student t test; ****P , .0001. (D) The body weight of treated mice was measured as an indicator of their overall condition. The change in
body weight compared with the day of T-cell injection for each treatment group is depicted. Data represent the mean of each group 6 SD. Results represent pooled data
from 5 animals.
leukemic blasts, but also on healthy HSCs or on normal myeloid
progenitor cells.13 CD70 has emerged as a promising target for
AML-directed therapies, because, under physiological conditions,
it is not expressed on healthy HSCs.13 Expression on normal tissue
is limited to a subset of immune cells such as activated T and
B lymphocytes and various types of antigen-presenting cells
Figure 4 (continued) CD70-CAR or NT T cells. (C) Quantitative analysis of bioluminescence signals for individual mice from each treatment group. (D) Kaplan-Meier survival plot
of mice treated with CD70-CAR or NT T cells. The log-rank (Mantel-Cox) test was used to perform statistical analyses of survival between the treatment groups. (E) The
experimental setup of the THP-1 xenograft model. After sublethal irradiation, NSG mice underwent injection of 1 3 106 luciferase-labeled THP-1 tumor cells. 4 days later, after
engraftment, the mice then received an injection of 5 3 106 CD70-CAR or NT T cells and were subsequently monitored by BLI. (F) Quantitative analysis of bioluminescence
signals for individual mice with THP-1 tumor cell engraftment and treatment with LF-28z-, CD27z-CAR, or NT T cells. (G) Cytotoxicity of LF-28z- and CD27z-CAR T cells Ts against
AML blasts from 3 patients with AML and various levels of CD70 expression was determined with a chromium-51 release assay. Data are the mean percentage of lysis 6 standard
deviation at an E:T target ratio of 40:1 from 3 different donors, with 3 technical replicates. *P , .05; **P , .01; ***P , .001, by unpaired Student t test.
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SAUER et al
A
B
8000
MFI (CD70 expression)
% CD70+ T cells
100
75
50
25
6000
4000
2000
0
0
d0 d1 d4 d8 d17
d0 d1 d4 d8 d17
Days after 1st stimulation
Days after 1st stimulation
C
FSC-A
NTC
250K
200K
150K
100K
50K
0
MVSTs
NTCs
0
IM-28z
103 104
250K
200K
150K
100K
50K
0
LF-28z
250K
200K
150K
100K
50K
0
MVSTs
CARTs
103 104
0
CARTs
CD27z
MVSTs
103 104
0
250K
200K
150K
100K
50K
0
CARTs
0
MVSTs
103 104
CellTrace Violet®
MVSTs
**
***
***
ns
105
ns
106
*
105
F
300
# of colonies
ns
100
0
40
30
20
10
N
T
IM C
-2
8
LF z
-2
8
CD z
27
z
****
****
200
ns
CBU donor #2
CBU donor #1
300
50
0
N
T
IM C
-2
8
LF z
-2
8
CD z
27
z
N
T
IM C
-2
8
LF z
-2
8
CD z
27
z
104
ns
200
100
ed
ia
N
TC
LF
-2
8z
CD CD
2
33 7z
CA
RT
m
ed
ia
N
TC
LF
-2
8z
CD CD
33 27z
CA
RT
0
m
# of colonies
E
NTCs/CARTs
107
Cell number
Cell number
106
% IFN-g+/TNF-a+ MVSTs
D
Figure 6. CD70-CAR T cells eliminate multivirus-specific T cells, but spare normal HSCs. (A-B) CD70 expression on MVSTs was determined by flow cytometry at different time
points after stimulation. Bar graphs representing the percentage of CD70-positive cells (A) and the mean fluorescence intensity (MFI) of CD70 expression of MVSTs (B) at the
indicated time points. CellTrace Violet–labeled MVSTs were cocultured with autologous CD70-CAR or NT T cells and harvested after 72 hours. The absolute cell count of both
T-cell populations was determined by flow cytometry with CountBright counting beads. (C) Representative dot plots for the different T-cell groups are shown, in which
autologous MVSTs can be distinguished from CAR or NT T cells by their CellTrace Violet labeling. (D) The total number of MVTSs (left graph) and CAR/NT T cells (right graph)
after 3 days of coculture. (E) The percentage of MVSTs with intracellular IFN-g and TNF-a expression after 3 days of coculture with CD70-CAR or NT T cells, followed by
stimulation with EBV-, adenovirus-, and CMV-specific pepmixes. Data are the mean 6 standard deviation (SD) of results from 4 different donors. (F) CD70-CAR, CD33-CAR, and
NT T cells were cocultured with normal CD34-positive HSCs for 6 hours, and the cells were seeded in a standardized medium for CFU assays. The total number of colonies after
the first plating were determined after 2 weeks of incubation. Data are the mean number of colonies formed by HSCs from 2 different donors after incubation with CAR or NT
T cells from 3 different donors. The error bars indicate the SD. Two independent investigators counted colonies from 2 technical replicates for each condition. *P , .05; **P , .01;
***P , .001; ****P , .0001; ns, not significant, by unpaired Student t test.
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29 JULY 2021 | VOLUME 138, NUMBER 4 327
(eg, epithelial and dendritic cells in the thymic medulla). CD70
provides costimulation to T cells through binding to its ligand
CD27.31,32 Both, CD70 and CD27 are frequently coexpressed in
myeloid leukemia blasts, and their interaction induces a stem
cell–like phenotype14 that is associated with impaired survival
of patients with AML.33 In our study, we demonstrated unequivocally, in a large series of primary AML bone marrow
samples, that CD70 was consistently overexpressed, extending
studies conducted by other investigators.13,14
Most CD70-targeted immunotherapies so far have focused
on monoclonal antibodies, which have been evaluated as
monotherapy34,35 or are being evaluated in combination with
other therapeutic agents (studies registered at www.clinicaltrials.
gov as #NCT04023526, #NCT04150887, and #NCT04241549).
Recently, Riether et al15 reported very promising results from an
early-phase clinical trial that investigated the combination of
cusatuzumab, a humanized monoclonal CD70-specific antibody
in combination with azacytidine for the treatment of patients with
AML. However, immunotherapy with monoclonal antibodies has
several limitations. The injection of large amounts of recombinant antibody is frequently necessary to achieve sufficient drug
levels in the serum and is associated with significant costs. In
addition, because of the limited persistence, continuous or repeated infusions of the monoclonal antibodies are necessary for
the duration of the treatment. The adoptive transfer of CARmodified T cells has the potential to overcome these limitations.
In this study, we preclinically evaluated a CAR T-cell–based
approach for the treatment of CD70-positive AML. We
designed a panel of scFv-based CAR T cells with different hinges
(IM, LF) and costimulatory molecules (CD27, CD28, and 4-1BB)
and 1 CAR based on the CD70 receptor CD27.18 The different
CAR constructs produced striking differences in the baseline
characteristics of the transduced cells with regard to viability,
phenotype, and ability to proliferate. Specifically, expression of
IM-based CAR T cells resulted in decreased T-cell viability and
terminal differentiation, in particular when paired with the 4-1BB
signaling domain. These findings suggest the presence of some
baseline signaling in our designed CAR T cells, also known as tonic
signaling, that depends on the structural composition of our
designed CAR T cells. In this regard, others have reported that the
4-1BB costimulatory domain and structural components of the
CAR, such as spacers, can modulate baseline signaling of CAR
T cells.36,37 The IM-based CAR T cells were also expressed on the
T-cell surface at higher levels than LF-based CAR T cells, which
most likely contributed to baseline signaling as reported by
others.36,38
LF-BBz- and CD27z-CAR T cells had superior effector function in
a repeat stimulation assays as judged by their sustained cytolytic
activity and ability to proliferate. Phenotypic analysis revealed
that CD27z-CAR T cells expressed proteins associated with
T-cell exhaustion (LAG-3, TIM-3, and PD-1) at significantly lower
levels after the third stimulation than T cells expressing all other
CAR T cells, most likely because the CD27z-CAR T cells induced
less differentiation than scFv-based CAR T cells with CD28 and
4-1BB costimulatory domains. Of interest, LF-27z-CAR T cells,
which, before antigen stimulation, had an indistinguishable
phenotype to CD27z-CAR T cells, also expressed higher levels
of LAG-3, TIM-3, and PD-1 after the third stimulation. Thus,
whereas CD27 costimulation has been shown to improve CAR
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29 JULY 2021 | VOLUME 138, NUMBER 4
T-cell function,39 in our system, CD27 costimulation by itself was
insufficient to prevent T-cell exhaustion.
In vivo, only LF-28z- and CD27z-CAR T cells had significant
antitumor activity in 2 different AML xenograft models. Although
this finding was expected for CD27z-CAR T cells based on our
coculture assay, which mimics chronic antigen exposure, it was
unexpected for CD70scFv-based CAR T cells because LF-BBzCAR T cells had a greater ability to sequentially kill tumor cells
than LF-28z-CAR T cells in vitro. However, LF-28z-CAR T cells
showed robust expansion after the first stimulation in vitro in
comparison with LF-BBz-CAR T cells, suggesting that initial CAR
T-cell expansion is important for tumor control in our AML xenograft models. Although LF-28z- and CD27z-CAR T cells had
similar anti-AML activity, the CD27z-CAR T cells induced greater
T-cell expansion and persistence in vivo than the LF-28z-CAR
T cells. This result is consistent with our in vitro repeat stimulation
assay and findings by others that CD28-based CAR T cells have
limited persistence in preclinical xenograft models.40 Several
groups have generated CD70-CAR T cells, using CD27 as the
antigen recognition domain to target CD70-positive solid tumors, and have shown significant antitumor activity in preclinical
models.18,41-43 To our knowledge, our study is the first to systematically compare the effector function of CD70scFv- and
CD27-based CAR T cells. In addition, it highlights that it is critical
to perform a comprehensive in vitro and in vivo analysis.
CD70-CAR T cells did not recognize and eliminate normal HSCs
in CFU assays, suggesting a clear advantage over CD33- and
CD123-CAR T cells, which produce significant on-target/offtumor toxicity to HSCs and myeloid progenitor cells.44,45 However, we observed that CD70-CAR T cells recognized activated
CD70 expressing MVSTs in vitro. Even though it is unclear at this
time whether this finding indicates that targeting CD70 would
put patients at higher risk for infections caused by T-cell–specific
immune defects, it has been shown that mice infused with
murine CD70-CAR T cells could still mount antigen-specific
T-cell responses,43 and treatment with the CD70 monoclonal
antibody ARGX-110 was well tolerated in a cohort of 26 heavily
pretreated cancer patients, without infectious complications.34
Of note, another T-cell activation marker, CD30, is also expressed on activated VSTs; however, patients receiving CD30CAR T cells for the treatment of CD30-positive lymphomas are
not at higher risk for viral infections.46 A clinical study (registered
on www.clinicaltrials.gov as #NCT02830724) with CD70-CAR
T cells for solid tumors is already accruing patients, but safety
data from this trial has not yet been published. Nevertheless,
based on our findings, we believe that monitoring T-cell–specific
immune responses in early-phase clinical studies with CD70-CAR
T cells is advisable. In addition, CD70 expression on activated
T cells may cause fratricide of CD70-CAR T cells, thereby potentially interfering with the production of CAR T cells ex vivo and/
or their in-patient expansion after antigen stimulation by tumor
cells. However, as we were able to detect robust proliferation of
CD27z-CAR T cells in the murine xenograft model, our data
suggest that decreased viability and expansion capacity of some
CD70-CAR T-cell products were attributable rather to differences
in the structural composition of the CAR than to fratricide induced
by CD70 expression of CAR T cells.
In summary, CD70 is a promising target antigen for CAR T-cell
therapy for AML. We designed a panel of CD70 CAR T cells,
SAUER et al
and T cells expressing CAR T cells based on the CD70 receptor
(CD27z-CAR T cells) emerged as the most beneficial CAR T-cell
product, as shown by their anti-AML activity and ability to expand
and persist. Thus, our results warrant future early-phase clinical
testing of CD27z-CAR T cells in patients with CD70-positive AML.
Acknowledgments
(DFG), Deutsche Krebshilfe, German Ministry of Education and Science
(BMBF), Wilhelm-Sander-Stiftung, Jose-Carreras-Stiftung, and Bayer; and
has served on the DSMB/SAB of Pfizer and Janssen-Cilag. M.S. has
received research funding from Apogenix, Hexal, and Novartis; has
served on the DSMB/SAB of MSD; is a cofounder of and shareholder in
Tolerogenixx; and has received financial support of educational activities
and conference attendance from Kite. C.S. has received reimbursement
for travel, accommodations, and expenses from Celgene, PharmaMar,
and Pfizer and has served on the DSMB/SAB of AbbVie, Jazz Pharmaceuticals,
Pfizer, Novartis, and Takeda. The remaining authors declare no competing financial interests.
This work was supported by National Institutes of Health, National
Cancer Institute grants P50 CA126752 and P01 CA094237 (C.M.R.),
Leukemia and Lymphoma Society grant 7001-19 (C.M.R.), and Cancer
Prevention and Research Institute of Texas grant RP160283 (Baylor
College of Medicine Comprehensive Cancer Training Program) (S.S.).
ORCID profiles: T.S., 0000-0001-5412-324X; B.O., 0000-0002-14795305; L.A., 0000-0003-2502-9910; S.G., 0000-0003-3991-7468.
Authorship
Correspondence: Tim Sauer, Department of Hematology and Oncology,
University Hospital of Heidelberg, Im Neuenheimer Feld 410, 69120
Heidelberg, Germany; e-mail: tim.sauer@med.uni-heidelberg.de.
Contribution: T.S., K.P., S.S., D.S., Q.C., L.A., and C.S. performed the
experiments; T.S., S.G., and C.M.R. analyzed and interpreted the data;
T.S., S.G., and C.M.R. designed the research and wrote the paper; and
B.O., M.S., and C.M.-T. contributed to the interpretation of the results.
Conflict-of-interest disclosure: T.S. has received support of educational
activities from Pfizer and AbbVie and has served on the Data and Safety
Monitoring Board and the Science Advisory Board (DSMB/SAB) of
Matterhorn Biosciences AG and Takeda. C.M.R. holds patents and patent
applications in the fields of T-cell and gene therapy for cancer; has received research support from TESSA Therapeutics; and has served on the
DSMB/SAB of TESSA Therapeutics, CellGenix, and Marker Therapeutics.
S.G. holds patents and patent applications in the fields of T-cell and gene
therapy for cancer; has received research support from TESSA Therapeutics; has served on the DSMB/SAB of Immatics and Tidal; and
has been a consultant within the past 2 years for Merck and ViraCyte.
C.M.-T. has received grants and/or provisions of investigational medicinal products (IMPs) from Pfizer, Daiichi Sankyo, and BiolineRx; has
received research funding from Deutsche Forschungsgemeinschaft
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Submitted 16 July 2020; accepted 5 March 2021; prepublished online on
Blood First Edition 15 March 2021. DOI 10.1182/blood.2020008221.
Original data are available by e-mail request to the corresponding
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