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Hours Post Cell Injection - obsidiantx.com · Dhruv Sethi, Kaylee Spano, Dexue Sun, Karen Tran,...
Transcript of Hours Post Cell Injection - obsidiantx.com · Dhruv Sethi, Kaylee Spano, Dexue Sun, Karen Tran,...
Obsidian Therapeutics, Inc. - 1030 Massachusetts Avenue, Suite 400 - Cambridge MA 0213 - [email protected]
Drug Dose
Regulation of CD40 Ligand Transgene
Expression in Human CAR-T Cells Using
FDA Approved DrugsElizabeth Weisman, Nathaniel Bagge, Emily Brideau, Kutlu Elpek, Michelle Fleury, Scott Heller, Christopher Reardon, Michael Schebesta,
Dhruv Sethi, Kaylee Spano, Dexue Sun, Karen Tran, Michael Briskin, Jennifer L. Gori, Celeste Richardson, Vipin Suri, and Steven Shamah
ABSTRACTChimeric antigen receptor modified T cells (CAR-T) have shown clinical efficacy in the treatment of
B cell malignancies and multiple myeloma. Several challenges restrict their application across
hematologic malignancies and solid tumors, including: limited CAR-T cell expansion and
persistence, tumor microenvironment-induced immunosuppression, and antigen negative tumor
escape. Cluster of Differentiation 40 Ligand (CD40L), a tumor necrosis factor superfamily member
transiently expressed on activated CD4 T cells, promotes dendritic cell (DC) licensing and activation
through interaction with the CD40 receptor. Co-expression of engineered CD40L in CAR-T cells has
the potential to reduce antigen-negative tumor escape even when native CD40L is downregulated,
thereby increasing antitumor efficacy. The cytokine program associated with CD40L-mediated DC
activation could also improve T cell expansion and activity. However, activation of the CD40 pathway
using agonistic antibodies causes systemic immune activation that has been associated with
adverse clinical events, thus limiting therapeutic application. We therefore hypothesized that precise
and titratable regulation of CD40L would allow for its safe inclusion in CAR-T cell therapy, thus
empowering the next generation of potent cellular immunotherapies.
To enable regulation of CD40L, we applied drug responsive domain (DRD) technology which utilizes
human protein domains that are inherently unstable in the cell but are reversibly stabilized when
bound to FDA-approved small molecule ligands. Fusion of transgenes to a DRD confers ligand-
dependent, reversible regulation to any protein of interest. We therefore fused human CD40L to a
DRD derived from the E. coli dihydrofolate reductase (ecDHFR) which can be regulated by the
clinically-approved antibiotic trimethoprim (TMP). We evaluated CD40L expression and in the
absence of ligand, the CD40L-DRD fusion was expressed at very low levels in transduced T cells.
Exposure to TMP increased CD40L expression in T cells in a dose-dependent manner. To test the
activity of CD40L-DRD, we incubated transduced Jurkat T cells with a reporter cell line that reads
out CD40 receptor activation. Addition of TMP increased CD40 activation to similar levels seen in
cells constitutively expressing CD40L. To evaluate the effect of regulated CD40L expression on DC
activation, monocyte derived human DCs were exposed to control or CD40L-DRD expressing T
cells. After TMP treatment, the levels of inflammatory cytokines IL12, TNFa, and IFNg were elevated
in co-cultures of DC and CD40L-DRD T cells compared to co-cultures of DCs and control T cells.
To determine the effect of CD40L on CAR-T activity in vivo, T cells expressing CD40L and CD19-
targeting CAR were infused into CD19+ Nalm6 tumor-bearing mice. Increased tumor regression was
seen in mice that received T cells co-expressing CD40L with CD19-targeting CAR compared to CAR
alone. Studies are underway to evaluate the effect of regulated CD40L expression on CAR-T cell
anti-tumor efficacy in vivo.
These findings indicate that CD40L can be regulated using DRDs and FDA approved small molecule
ligands, and that regulated CD40L transgene expression by human T cells promotes DC activation
and increases CAR-T antitumor activity. Regulated CD40L can be applied to CAR-T therapy to
enhance immunotherapy potency by increasing T cell expansion, promoting DC activation, and
inducing further epitope spreading.
•Expression of constitutive CD40L in human T cells enhances CAR efficacy
and dendritic cell activation in vivo
•Drug responsive domains enable regulation of CD40L expression using
FDA-approved small molecule drugs at clinically achievable
concentrations
•Regulated CD40L in T cells activate dendritic cells to produce IL12
TM
Obsidian Therapeutics, 1030 Massachusetts Avenue,
Suite 400, Cambridge MA 02138
SUMMARY
Figure 8: DRD fused CD40L in T cells supports pharmacological regulation of DC
activation in vitro
In vitro coculture for 48h
Human monocyte derived
Dendritic Cells
(GM-CSF and IL4 for 5 days)
Human T cells
transduced with
regulated CD40L
Ratio of
1 : 10
Secreted IL12
+/- ligand
CD4+ CD8+In vitro
Empty LV
ecDHFR-CD40L + Vehicle
CD40L LV
ecDHFR- CD40L + TMP (10 mM)
In vivo
A) Pre infusion, T cells were transduced with lentivirus expressing constitutive or E. coli DHFR regulated CD40L. Two days later, cells were treated
with vehicle or 10 mM TMP for 24h after which they were analyzed for CD40L surface expression. B) The same T cells were expanded in vitro for
10 days before infusion into NSG mice (n=4/group). Two days after infusion, animals were dosed orally 3x at the indicated times with 500 mg/kg
TMP (▼). Blood samples taken prior to dosing or 2, 6, 10 and 24 hours post-dosing were analyzed for CD40L surface expression on human T cells.
Surface expression reached a maximum at 10 hours after the first dose.
Figure 7: Trimethoprim induces regulatable expression of DRD fused CD40L in vivo
Figure 9: Regulation of CD40L with a human DRD from carbonic anhydrase 2
To study the kinetics of regulated surface expression, activated T cells were stably transduced with CA2 regulated CD40L. A) Cells were treated
with various doses of acetazolamide (ACZ) or DMSO and fixed at the indicated timepoints. Cells were stained, and surface CD40L was measured
using FACS analysis. Expression reached its peak at 24 hours with highest doses expressing higher than constitutive levels. B) Dose response
curves of CA2 regulated CD40L with ACZ blotted as median fluorescent intensity or gated percent positive. The indicated area is an approximate
Cmax of ACZ concentration in the plasma of humans from various clinical studies.
BA
BA
8:
0 2 4 6 8
0
2 0 0 0
4 0 0 0
6 0 0 0
8 0 0 0
1 0 2 0 3 0 4 0 5 0 6 0
T im e (h rs )
CD
40
L M
FI
E m p ty L V
C D 4 0 L L V
C A 2 -C D 4 0 L + D M S O
C A 2 -C D 4 0 L + A C Z
ON Kinetics (CD4+CD8+) Dose Response
Figure 4: Addition of a drug responsive domain (DRD) allows for regulation of protein
expression using FDA approved small molecules
Our technology provides control over protein expression via the administration of safe, FDA-approved small molecule drugs. We incorporate protein
units called Drug Responsive Domains (DRDs) into the transgene structure. DRDs are expressed as unfolded units that confer rapid degradation to
fused proteins through the cell’s proteasome machinery. Binding of the small molecule ligand to the DRD stabilizes the complex enabling the
expression and function of the target gene product. Importantly, the surface expression is titratable dependent on the concentration of dosed drug
providing fine-tuned control over protein function.
Figure 5: Five drug responsive domains regulate CD40L with FDA approved drugs
Drug responsive domains were made by mutating the proteins E.coli and human dihydrofolate reductase (DHFR), estrogen receptor (ER),
phosphodiesterase isozyme 5 (PDE5), and carbonic anhydrase 2 (CA2). To test regulation, activated T cells were transduced with lentivirus
expressing DRD regulated CD40L. Two days later, cells were treated with vehicle or ligand for 24h after which they were analyzed for CD40L
surface expression. Note the endogenous expression of CD40L in activated CD4-cells (grey). Regulated expression with all destabilizing domains
significantly enhanced CD40L expression beyond endogenous levels. Both E.coli DHFR and CA2 drug responsive domains show levels close to
constitutive expression with clinically relevant ligand doses.
Figure 6: DRD fused CD40L induces expression with rapid off kinetics after drug washout
ON Kinetics OFF Kinetics
To study the kinetics of regulated surface expression, Jurkat cells were stably transduced with E.coli DHFR regulated CD40L. A) For ON kinetics
measurements, cells were incubated with TMP for the indicated duration of time before analysis for CD40L expression by FACS. B) For OFF
kinetics, cells were incubated with TMP or vehicle for 24h followed by extensive PBS washing and fixation of the cells at the indicated timepoints
after washout. CD40L surface expression of the cells was determined by FACS. Interestingly, surface expression increases moderately after TMP
addition which could be a consequence of the necessary trimerization for proper surface trafficking of CD40L. In contrast, after washout of TMP,
cells quickly lost surface expression.
BA
Healthy donor derived human T cells were activated, virally transduced with CD40L or ecDHFR-CD40L and expanded. ) In addition, allogeneic
human monocytes were differentiated with IL4 and GM-CSF for 5 days before cryopreservation. Cells were cryopreserved and freshly thawed for
co-culture experiment. For co-cultures both cell types were freshly thawed and incubated together at a 10:1 ratio (T cell:moDC) for 2 days before
secreted IL12 was analyzed. For E. coli DHFR regulation, TMP was added during the co-culture period.
Activation of CD40+ dendritic cells
promotes epitope spreading
Figure 1: CD40 Ligand can help to overcome challenges of adoptive cell therapies
Reverse signaling and cytokine
production enhances antigen-dependent
T cell expansion
Repolarization of CD40+ macrophages in
tumor microenvironment to
proinflammatory state
CD40
CD40
Figure 2: Engineered CD40L expression on T cells activates dendritic cells in vivo
CD40L
CD4+
CD8+
Human monocyte derived
dendritic cells
(GM-CSF and IL4 for 5 days)
Human T cells
transduced with
constitutive CD40L
sequential IP
injections into NSG
mice
Ratio of
1 : 5
Plasma IL12 secreted from activated DCs
CD40L expression in
transduced human T cellsT cells engineered to overexpress CD40L activate dendritic cells to secrete IL12
Figure 3: CD40L expression enhances CD19-targeted CAR-T cell anti-tumor efficacy in vivo
A) Activated T cells were transduced with lentivirus expressing CD40L, CD19-targeted CAR, or CD40L with CD19-targeted CAR. Cells were grown
for 2 days and CD40L and CD19 expression analyzed by flow cytometry. B) CD19+ luciferase+ Nalm6 tumor-bearing NSG mice were injected with
transduced human T cells 7 days post tumor implantation (n=8/group). Tumor burden was analyzed by bioluminescence imaging (photons/second,
p/s). In this model, tumors are regressed when total flux ≦10 6.
CD
19-C
AR
Empty LV CD40L CD19-CARCD19-CAR-
P2A-CD40L
CD40L
10 20 30105
106
107
108
109
1010
1011
Days Post Nalm6luc Tumor Implant
Tum
or
burd
en
(Tota
l F
lux [p/s
])
Empty Vector
CD40L
CD19-CAR
CD19-CAR P2A CD40L
CD40L armored CAR-T cells regress tumors at
suboptimal CAR-T cell doseTransduced T cells co-express CD19-CAR and CD40L BA
A) Healthy donor human T cells were activated, transduced with CD40L-expressing lentivirus vector (LV), expanded,
frozen, and thawed and CD40L levels analyzed by flow cytometry. B) Allogeneic human monocytes were
differentiated into moDCs. For in vivo DC activation experiment, both cell types were freshly thawed and separately
injected intraperitonially at a 5:1 ratio (T cell:moDCs) into NSG mice. Plasma was collected at indicated timepoints
post cell infusion and analyzed by miso scale discovery (MSD) for interleukin 12 (IL12).
BA
Shedding
Site
N Terminus
Intracellular
signaling domain
TNF Homology
Domain
(CD40 receptor
binding)
Insufficient
T cell expansion
Immunosuppression
Tumor antigen
escape.
CAR or
TCR
CD40L
Challenges with
CAR-T or TCRCD40 pathway activation BA
Extended activation of the CD40 pathway by agonistic
antibodies has been associated with adverse clinical events.
Fusion to Obsidian’s drug responsive domain (DRD)
technology allows for liganded control of protein expression.
0 240
10
20
30
hours after first dose
%C
D40
L+
2 6 10 12
TMP TMP TMP
Empty LV
E.coli DHFR-CD40L + vehicle
E.coli DHFR-CD40L + 3x TMP (500mg/kg)
CD
40L M
FI
CD40L
E.Coli DHFR CD40L
+ Trimethoprim
ER CD40L
+ Bazedoxifene
PDE5 CD40L
+ Vardenafil
CA2 CD40L
+ Acetazolamide
1000
2000
3000
4000Vehicle treated for 24 hour before washout
TMP (50 µM) treated for 24 hour before washout
0 3 6 9 24
CD
40L M
FI
Hours post TMP washoutHours post TMP addition
CD
40
L M
FI
1000
2000
3000
4000
0 3 6 9 24
Vehicle
TMP (50 µM)
vehicle 0.1 0.5 2 10
+ DD-CD40L T cells
T M P ( m M)
+ EV T
cells
+ CD40L T
cells
0
20
40
60
80
Secreted IL-12
IL1
2 (
pg
/mL)
Secreted IL12
IL12 (
pg/m
L)
TMP (µM) .
Vehicle 0.1 0.5 2 100
-
CD40L
DRD
0.0
0.5
1.0
10
20
30
40
CAR/CD40L dependent T-cell expansion(at 14 days post T cell infusion)
% o
f hum
an c
ells
in the b
lood
EV T cell CAR+ CAR-P2A
CD40L
% h
um
an c
ells
in b
lood
Human Cmax (500 mg dose)
Isotype
Empty LV
CD40L LV
0 10 20 30 400
100
200
300
Hours Post Cell Injection
IL1
2 (
pg
/mL)
Naive mouse
EV T-cells only
CD40L T-cells only
moDCs + EV T-cells
moDCs + CD40L T-cells
(EV=empty vector)
Plasma IL12
Naïve Mouse
Empty LV T cells alone
CD40L LV T cells alone
moDCs + EV T cells
moDCs + CD40L T cells
IL12 (
pg
/mL
)
Hours Post Cell Injection 0 10 20 30 40
0
100
200
300
Hours Post Cell Injection
IL1
2 (
pg
/mL)
Naive mouse
EV T-cells only
CD40L T-cells only
moDCs + EV T-cells
moDCs + CD40L T-cells
(EV=empty vector)
% P
osit
ive
CD4+
CD8+
E.Coli DHFR CD40L + Trimethoprim
CD40L
+ Trimethoprim
hDHFR CD40L
EV
DMSO
Drug
Days Post Tumor Implant
T cell expansion at
14 days post infusion
%
CD
40L
Po
sit
ive
Empty LV
CD40L LV
CD19 CAR LV
CD19-CAR-P2A-CD40L LV
E.Coli DHFR CD40L + Trimethoprim
E.Coli DHFR CD40L + Trimethoprim
CA2 CD40L + Acetazolamide
QD BID QD BID
CD4+
CD8+
CD4+
CD8+
CD40L
4-1BB CD3zCD19 scFv P2A CD40L
CD40 LV
CD19 CAR LV
CD19 CAR-P2A-CD40L LV
4-1BB CD3zCD19 scFv
DRD
CD40L