LB-184
Dual-switch HER2 CAR-T cells:
Small molecule-regulated GO and STOP switches to target solid cancer in vivo
Mylinh T Duong, Matthew R Collinson-Pautz, Eva Morschl, Mary Brandt, Ming Zhang, Kevin M Slawin, Aaron E Foster, J Henri Bayle*, David M Spencer*
Bellicum Pharmaceuticals, Houston, TX
Background
Results
Retrovirus and transduction: T cells were activated with anti-CD3/28 antibodies and transduced with a
bicistronic retrovirus encoding tandem Rim-binding domains (FKBP12v36), cloned in-frame with MyD88 and
CD40 cytoplasmic signaling molecules, and a first generation CAR targeting HER2 (SFG-iMC.2A-HER2.ζ). To
generate Dual-switch HER2 CAR-T cells, the cells were co-transduced with retrovirus encoding the RapaCIDe
plasmid, which contains tandem FRB and FKBP domains, cloned in-frame with a truncated caspase-9 gene
followed by a CD19 transmembrane domain (SFG-iRC9.2A-CD19).
In vitro assays: (1) The effects of iMC costimulation on HER2-targeted CARs were assessed in coculture
assays with HER2+ cancer cell lines (HPAC data shown; OE19 and SKOV3 data not shown) ± Rim using the
IncuCyte® live cell imaging system. T cells were labeled with retrovirus encoding GFPFluc gene. IL-2 release
was examined by ELISA from coculture supernatants 48h after set up. (2) Apoptosis assays were performed
in the IncuCyte ± Rap and in the presence of IncuCyte Caspase 3/7 Reagent. After 24 hours of drug
stimulation, flow cytometry was performed with AnnexinV and PI.
In vivo assays: (1) In vivo efficacy of Dual-switch HER2 CAR-modified T cells was assessed using an immunedeficient NSG tumor xenograft model. GFPFluc-expressing HER2+ OE19 tumor cells were injected s.c.
Following engraftment, modified T cells were administered i.v. and then immediately with i.p. injections of
Rim (1 mg/kg) or vehicle and continued weekly thereafter. Animals were evaluated for OE19-GFPFluc tumor
burden and T cell expansion (Orange Nano Lantern-renilla luciferase or ONLRluc) on a weekly basis using IVIS
bioluminescent imaging (BLI). (2) To test the apoptotic switch, CaspaCIDe™ or RapaCIDe-modified T cells (colabeled with GFPFluc) were injected i.v. followed by 1 dose of Rim/Rap i.p. 24 hours later. BLI was assessed at
various time points.
RapaCIDe-T Dual-switch HER2 CAR-T
Rim
Veh
Rim
-1
7
6
14
13
24
†
†
28
† † † † †
35
† † † † †
10
† † † † †
34
† † † † †
41
† † † † †
† † † † †
54
† † † † †
53
† † † † †
A
CaspaCIDe-T
Veh
Rim
Figure 2. Activated T cells were co-transduced with retroviruses
encoding the RapaCIDe vector ± HER2 GoCAR vector and the
ONLRluc vector. NSG mice were engrafted with 2x106 OE19-GFPFluc
cells s.c. for 4 days followed by infusion of 2.5x106 CD19-selected T cells i.v. Rimiducid (1 mg/kg)
or placebo were given i.p. on day 0 and weekly thereafter. (A & D) OE19-GFPFluc growth was
measured using IVIS BLI with luciferin substrate. (B & E) T cell expansion was measured using IVIS
BLI with coelenterazine substrate. (C) Caliper measurement of the tumors was performed weekly.
NT
GoCAR-T
C
B
RapaCIDe-T
Rap
Veh
Rim
Rap
0
5
24
D
Drug-specific elimination of T cells
0.0
E
PI
Activation of the RapaCIDe switch eliminates T cells in a
dose-dependent manner in vivo.
*
Veh
Rim
Rap
0.5
46
Dual-switch
HER2 CAR-T
Figure 3. PBMCs from 2 donors were activated and
transduced with retrovirus encoding the HER2 GoCAR-T
vector or co-transduced with the RapaCIDe vector for
the Dual-switch HER2 CAR-T group. Eleven days posttransduction, GoCAR-T cells were selected with CD34
microbeads and Dual-switch HER2 CAR-T cells were
selected with CD19 microbeads. (A) On day 13, T cells
were seeded on 96-well plates with 0, 1, and 10 nM
rapamycin and in the presence of 2 μM caspase 3/7
green reagent to monitor real-time caspase cleavage. (B
& C) After 24 hours, cells were stained with annexin V
and propidium iodide for flow cytometry detection of
apoptotic cells. *p-value < 0.05, 2-way ANOVA.
AnnV
Casp. 3/7 activation
Dual-switch
HER2 CAR-T
Time (24h)
1.0
27
B
Rap (nM)
GoCAR-T
*
† † † † †
D
1
Non-transduced
C
20
47
C
0
CaspaCIDe-T
RapaCIDe-T
Avg Radiance (p/s/cm2/sr)
T cell IVIS
Dual-switch HER2 CAR-T
Veh
Rim
A
E
Splenocyte CD3+/CD19+ (MFI)
Methods
RapaCIDe-T
Rim
2
• The allele specificity of rimiducid to the FKBPV mutant, and not to wild-type
FKBP, prevents cross-talk between the ON and OFF switches.
• While rapamycin is mechanistically a heterodimerizer, here it functions as a
homodimerizer to activate the RapaCIDe switch.
B
OE19 Tumor IVIS
A
iRC9.2A-∆CD19
RapaCIDe
Activation of the RapaCIDe switch quickly and efficiently
eliminates Dual-switch HER2 CAR-T cells in vitro.
Hours post drug
treatment
IL-2
(pg/ml)
HPAC-RFP
T CELLS-GFP
CELL DEATH
Activation of the iMC switch enhances tumor killing and
Dual-switch HER2 CAR-T cell proliferation in vivo.
Avg. Radiance (p/s/cm /sr)
Titration of rapamycin allows partial T cell iMC.2A-scFv.Q.ζ
GoCAR
elimination, preserving CAR-T function.
Figure 1. PBMCs from 2
donors were activated and
co-transduced
with
retroviruses encoding the
HER2-GoCAR-T
vectors,
the RapaCIDe vector, and
the GFPFluc vector. Six
days post-transduction, T
cells were cocultured at
decreasing E/T ratios with
HPAC-RFP cells ± Rim and
placed in the IncuCyte to
monitor T cell-GFP and
HPAC-RFP growth. (A) Two
days post-seeding, culture
supernatants
were
analyzed for IL-2 release
by ELISA. (B) Number of
HPAC-RFP cells and (C)
total green fluorescence
intensity of T cell-GFP
were analyzed using the
Incucyte basic analyzer
software at day 7.
E/T ratio
(wt)
Days post T cell injection
CELL ACTIVATION
FRB
FKBP
2
o
Rapid and efficient clearance of T cells
follows rapamycin administration.
+
Avg. Radiance (p/s/cm /sr)
o
FKBPV
FKBPV
Days post T cell injection
• APOPTOTIC SWITCH
ζ
C C
2
MM
Tumor size (mm )
Full activation and tumor cell killing
requires a second, target-specific CD3ζ
signal.
iRC9
o
Q
ΔC9
ΔC9
ΔC9
“On demand” co-stimulation via rimiducid
administration enhances cell proliferation
and activation.
iMC
o
Rimiducid (1 nM)
B
C
Rap
Rim
Rimiducid (0 nM)
A
(#cells/well)
Technology
Activation of the iMC switch enhances tumor killing and
Dual-switch HER2 CAR-T cell proliferation in vitro
(fluorescence intensity)
• While chimeric antigen receptor (CAR)-T immunotherapies have shown remarkable
efficacy against leukemia and lymphoma, improved CAR-T efficacy and persistence
without compromising safety is likely required to treat most solid tumors.
• We present two independently regulated molecular switches that elicit specific and
rapid induction of cellular responses upon exposure to their cognate ligands.
• Cell activation is controlled by the homodimerizer rimiducid (Rim) that triggers
signaling cascades downstream of MyD88 and CD40 via dimerization of an
engineered chimeric costimulatory protein termed iMC.
• The rapamycin (Rap)-controlled pro-apoptotic switch RapaCIDe™, or iRC9, is coexpressed and is also directed by drug-controlled protein homodimerization of
caspase-9 to mitigate possible toxicity from excessive CAR-T function.
• When combined with a first generation CAR, these two molecular switches allow
for specific and efficient regulation of engineered T cells.
• ACTIVATION SWITCH
*Corresponding authors – [email protected], [email protected]
Figure 4. The RapaCIDe switch is as efficient as the CaspaCIDe switch at activating apoptosis.
PBMCs from donor 676 were activated and transduced with retroviruses encoding the GFPFluc and
the CaspaCIDe or RapaCIDe vectors. NSG mice were injected i.v. with 1x107 T cells 24h before
treatment with rimiducid (5 mg/kg) or rapamycin (10 mg/kg) i.p. (A & B) BLI of cells was assessed at
-14, 0, 5, and 24 hours post-drug administration. (C & D) At 24h post-drug treatment, mice were
euthanized and spleens were collected for flow cytometry analysis with antibodies to hCD3, hCD19,
and mCD45. *p-value < 0.05, 2-way ANOVA. (E) Dose titration of rapamycin treatment in NSG mice
infused with 107 RapaCIDe-T cells at 24 hours post-drug.
Summary
• Dual-switch HER2 CAR-T, a novel platform combining a first-generation
CAR with independently regulated costimulatory and apoptotic signaling
elements, effectively controls tumor growth and T cell expansion and
elimination in vitro and in vivo.
• This dual-switch technology provides a user-controlled system for
managing persistence and safety of tumor antigen-specific CAR-T cells.