Regulation of CD40 Ligand Transgene TM

Obsidian Therapeutics, 1030 Massachusetts Avenue, Expression in Human CAR-T Cells Using Suite 400, Cambridge MA 02138 FDA Approved Drugs

Elizabeth 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 ABSTRACT Chimeric antigen modified T cells (CAR-T) have shown clinical efficacy in the treatment of Figure 5: Five drug responsive domains regulate CD40L with FDA approved drugs malignancies and . Several challenges restrict their application across E.Coli DHFR CD40L hDHFR CD40L ER CD40L PDE5 CD40L CA2 CD40L hematologic malignancies and solid tumors, including: limited CAR- expansion and persistence, tumor microenvironment-induced immunosuppression, and antigen negative tumor + Trimethoprim + Trimethoprim + Bazedoxifene + Vardenafil + Acetazolamide

escape. Cluster of Differentiation 40 Ligand (CD40L), a tumor necrosis factor superfamily member CD4+ 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 program associated with CD40L-mediated DC EV

activation could also improve T cell expansion and activity. However, activation of the CD40 pathway DMSO CD8+ using agonistic causes systemic immune activation that has been associated with Drug 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 .

To enable regulation of CD40L, we applied drug responsive domain (DRD) technology which utilizes human domains that are inherently unstable in the cell but are reversibly stabilized when CD40L

bound to FDA-approved small molecule ligands. Fusion of transgenes to a DRD confers ligand- Drug responsive domains were made by mutating the E.coli and human dihydrofolate reductase (DHFR), estrogen receptor (ER), dependent, reversible regulation to any protein of interest. We therefore fused human CD40L to a 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 DRD derived from the E. coli dihydrofolate reductase (ecDHFR) which can be regulated by the surface expression. Note the endogenous expression of CD40L in activated CD4-cells (grey). Regulated expression with all destabilizing domains clinically-approved antibiotic trimethoprim (TMP). We evaluated CD40L expression and in the significantly enhanced CD40L expression beyond endogenous levels. Both E.coli DHFR and CA2 drug responsive domains show levels close to absence of ligand, the CD40L-DRD fusion was expressed at very low levels in transduced T cells. constitutive expression with clinically relevant ligand doses. 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 Figure 6: DRD fused CD40L induces expression with rapid off kinetics after drug washout out CD40 receptor activation. Addition of TMP increased CD40 activation to similar levels seen in E.Coli DHFR CD40L + Trimethoprim 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 A ON Kinetics B OFF Kinetics 4000 4000 cells. After TMP treatment, the levels of inflammatory IL12, TNFa, and IFNg were elevated Vehicle Vehicle treated for 24 hour before washout in co-cultures of DC and CD40L-DRD T cells compared to co-cultures of DCs and control T cells. TMP (50 µM) TMP (50 µM) treated for 24 hour before washout 3000 3000 To determine the effect of CD40L on CAR-T activity in vivo, T cells expressing CD40L and CD19- 2000

targeting CAR were infused into CD19+ Nalm6 tumor-bearing mice. Increased tumor regression was 2000

CD40L MFI CD40L CD40L MFI CD40L

seen in mice that received T cells co-expressing CD40L with CD19-targeting CAR compared to CAR MFI CD40L alone. Studies are underway to evaluate the effect of regulated CD40L expression on CAR-T cell 1000 1000 anti-tumor efficacy in vivo.

These findings indicate that CD40L can be regulated using DRDs and FDA approved small molecule 0 3 6 9 24 0 3 6 9 24 ligands, and that regulated CD40L transgene expression by human T cells promotes DC activation Hours post TMP addition Hours post TMP washout and increases CAR-T antitumor activity. Regulated CD40L can be applied to CAR-T therapy to To study the kinetics of regulated surface expression, Jurkat cells were stably transduced with E.coli DHFR regulated CD40L. A) For ON kinetics enhance potency by increasing T cell expansion, promoting DC activation, and 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 inducing further epitope spreading. 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. Figure 1: CD40 Ligand can help to overcome challenges of adoptive cell therapies Challenges with Figure 7: Trimethoprim induces regulatable expression of DRD fused CD40L in vivo A B CD40 pathway activation CAR-T or TCR Repolarization of CD40+ macrophages in E.Coli DHFR CD40L + Trimethoprim TNF Homology Domain CD40 tumor microenvironment to proinflammatory state In vitro In vivo Immunosuppression (CD40 receptor A CD4+ CD8+ B CD40L binding) TMP TMP TMP 30 Activation of CD40+ dendritic cells Shedding CD40 promotes epitope spreading

Tumor antigen Site +

L 20

escape 0

. 4

CAR or - D TCR Reverse signaling and cytokine C % 10 production enhances antigen-dependent Positive CD40L

N Terminus T cell expansion % Intracellular 0 signaling domain 0 2 6 10 12 24 CD40L hours after first dose Insufficient DRD Empty LV T cell expansion Extended activation of the CD40 pathway by agonistic Empty LV ecDHFR-CD40L + Vehicle E.coli DHFR-CD40L + vehicle antibodies has been associated with adverse clinical events. CD40L CD40L LV E.coli DHFR-CD40L + 3x TMP (500mg/kg) Fusion to Obsidian’s drug responsive domain (DRD) ecDHFR- CD40L + TMP (10 mM) technology allows for liganded control of protein expression. 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 Figure 2: Engineered CD40L expression on T cells activates dendritic cells in vivo 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. CD40L expression in T cells engineered to overexpress CD40L activate dendritic cells to secrete IL12 A transduced human T cells B Plasma IL12 Naive mouse + Human monocyte derived Human T cells 300 CD4 NaïveNaive mMouseouse EV T-cells only Figure 8: DRD fused CD40L in T cells supports pharmacological regulation of DC dendritic cells transduced with300 (GM-CSF and IL4 for 5 days) constitutive CD40L EmptyEV T-c eLVlls Ton cellslyCD4 alone0L T-cells only activation in vitro

CD40LCD40L TLV-c eTll scellsm oonDly Calones + EV T-cells E.Coli DHFR CD40L + Trimethoprim )

L 200 moDCsmoDCs + EEVV TmT- occellseDlClss + CD40L T-cells

) m

/ Secreted IL-12 L 200 moDCs + CD40L T cells

g moDCs + CD40L T-cells

m p

/mL) Secreted IL12

/ (

Human monocyte derived Human T cells

Ratio of g (EV=empty vector) 80

2

pg

p ( 1 Dendritic Cells transduced with (EV=empty vector)

1 : 5 L 100 2 Isotype I

(GM-CSF and IL4 for 5 days) regulated CD40L )

1 L

100 L I

Empty LV ( IL12

sequential IP 60 m

CD40L LV /

% % Positive CD8+ injections into NSG DRD g

CD40L /mL)

mice 0

p pg 0 10 20 30 40 ( 40 0 Plasma IL12 secreted from activated DCs 0 10 20 Hours P30ost Cell I4n0jection 2

Hours Post Cell Injection Ratio of 1 IL12 ( IL12 Hours Post Cell Injection L 1 : 10 I 20 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 0 differentiated into moDCs. For in vivo DC activation experiment, both cell types were freshly thawed and separately s ls ll vehicleVehicle 0.1 0.1 0.5 0.5 22 10010 l e injected intraperitonially at a 5:1 ratio (T cell:moDCs) into NSG mice. Plasma was collected at indicated timepoints e c In vitro coculture for 48h c TMPT M P (µM)( m M) . T T CD40L post cell infusion and analyzed by miso scale discovery (MSD) for interleukin 12 (IL12). L V 0 E 4 +/- ligand + D C + DD-CD40L T cells + Figure 3: CD40L expression enhances CD19-targeted CAR-T cell anti-tumor efficacy in vivo Secreted IL12

Healthy donor derived human T cells were activated, virally transduced with CD40L or ecDHFR-CD40L and expanded. ) In addition, allogeneic Transduced T cells co-express CD19-CAR and CD40L CD40L armored CAR-T cells regress tumors at A B suboptimal CAR-T cell dose 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 CD19-CAR- 1011 secreted IL12 was analyzed. For E. coli DHFR regulation, TMP was added during the co-culture period. Empty LV CD40L CD19-CAR Empty Vector P2A-CD40L Empty LV CD40L 1010 CD40L LV

CD19CD19- CCARAR LV

CAR

)

- ] n 9 CD19CD19--CCARAR P-P2A2A C-DCD40L40L LV

s Figure 9: Regulation of CD40L with a human DRD from carbonic anhydrase 2 e

/ 10

d

p

[

r

u x CAR/CD40L dependent T-cell expansion

CD19 T cell expansion at

u b

l 8

10 (at 14 days post T cell infusion) CA2 CD40L + Acetazolamide

r F

14 days post infusion

l

o

d

o

a o

t 40

l

m

b

o

CD40L u 7 + + e T 10 30 ON Kinetics (CD4 CD8 ) Dose Response

h A B

T (

t

n

i 20

CD40L s

CD40 LV l

l 8 0 0 0

6 e 10 Human Cmax (500 mg dose) c

10 n

a 1.0

CD19 scFv 4-1BB CD3z m

CD19 CAR LV u 0.5 6 0 0 0

h

I

5 f QD BID QD BID F

10 o 0.0

% human blood in cells M

CD19 CAR- % +

+ 10 20 30 EV T cell CAR+ CAR-P2A CD4 CD4 CD19 scFv 4-1BB CD3z P2A CD40L L 4 0 0 0 P2A-CD40L LV CD40L 0 + +

Days PDaysost N Postalm 6Tumorluc Tu Implantmor Implant 4 CD8 CD8

D C A) Activated T cells were transduced with lentivirus expressing CD40L, CD19-targeted CAR, or CD40L with CD19-targeted CAR. Cells were grown 2 0 0 0 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, 0 p/s). In this model, tumors are regressed when total flux ≦10 6. 0 2 4 6 8 1 0 2 0 3 0 4 0 5 0 6 0 T im e (h rs )

E m p ty L V Figure 4: Addition of a drug responsive domain (DRD) allows for regulation of protein C D 4 0 L L V C A 2 -C D 4 0 L + D M S O expression using FDA approved small molecules C A 2 -C D 4 0 L + A C Z 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.

SUMMARY •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 Drug Dose Our technology provides control over protein expression via the administration of safe, FDA-approved small molecule drugs. We incorporate protein concentrations 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 •Regulated CD40L in T cells activate dendritic cells to produce IL12 providing fine-tuned control over protein function.

Obsidian Therapeutics, Inc. - 1030 Massachusetts Avenue, Suite 400 - Cambridge MA 02138: - [email protected]