Obsidian Therapeutics, Inc. - 1030 Massachusetts Avenue, Suite 400 - Cambridge MA 0213 - info@obsidiantx.com

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