Selective delivery of exosome-mediated STING agonist to antigen presenting cells results in significantly improved potency and reduced toxicitySu Chul Jang1, Christine Sia1, Raymond J. Moniz1, Joyoti Dey2, Rane Harrison1, Nikki Ross1, Ke Xu1, Kevin Dooley1, Nuruddeen Lewis1, Marc Grenley2, Christine McCoy1, Agata Villiger-Oberbek1, Scott Estes1, Jorge Sanchez-Salazar1, Kyriakos Economides1, Sriram Sathyanarayanan1

1 Codiak Biosciences, 500 Technology Square 9th Floor, Cambridge, MA 02139; 2 Presage Biosciences, 530 Fairview Avenue North, Suite 1000, Seattle, WA 98109

AbstractEmerging research has established the role of exosomes as an efficient natural messenger system todeliver macromolecules between cells. We have leveraged this capacity to develop a novel,engineered exosome therapeutic, to selectively deliver agonists of the Stimulator of Interferon Gene(STING) pathway to tumor resident antigen presenting cells (APC). exoSTING is composed ofexosomes, which are molecularly engineered to over-express an exosomal membrane glycoprotein,and which are loaded ex vivo with a STING agonist (SA). In vitro assays with human PBMCs showedexoSTING enhanced the potency of dendritic cell and monocyte activation and IFNβ production by100-fold compared to comparable amounts of free SA. Although liposomal formulated SA alsoimproved potency, it resulted in dose-dependent loss of viability in APC. Intra-tumoral micro-dosingof exoSTING with the CIVO® platform demonstrated selective activation of pTBK1 and pIRF3 in APCresulting in superior IFNβ production compared to free SA. Anti-tumor activity of exoSTING and freeSA was compared in a checkpoint therapy refractory B16F10 tumor model. Intra-tumoral (IT)administration of exoSTING resulted in 500-fold enhancement in potency versus free SA, with dose-dependent anti-tumor activity resulting in tumor cures in 50% of the mice in the highest (0.2 µg)exoSTING dose cohort. exoSTING treated mice were refractory to re-challenge with B16F10demonstrating the presence of an immune memory response. IT administration of efficacious dosesof exoSTING stimulated robust IFNβ but did not result in systemic induction of inflammatory cytokinesas seen with an efficacious dose of free SA. exoSTING IT treatment induced IFNγ regulated genes,PD-L1 and chemokines responsible for T-cell recruitment in the tumor, resulting in significant systemicinduction of tumor antigen-specific T cell response. exoSTING affords selective agonism of the STINGpathway in tumor resident APC that results in improved potency, reduced systemic toxicity andenhanced T-cell responses, and highlights the potential of our exosome engineering technology asan impactful therapeutic platform.

Leveraging the exosome communication process in the tumor microenvironment

Figure 1. Cancer cells communicate naturally with immune cells via exosomes. Tumor derived exosomes can express PD-L1 on the surface and suppress T-cell function. Following chemo or radiation therapy double stranded tumor DNA can be delivered to APCs via exosomes and activate anti-tumor immune response.

• Exosomes engineered to over-express protein X (PrX), a surface glycoprotein

• Loaded with a stabilized CDN agonist

• Provides selective delivery to APCs

• Superior potency, Reduces toxicity to immune cells

• Activates a potent T-cell mediated anti-tumor immune response

• Intra-tumoral or catheter based administration

exoSTING: a precision engineered exosome therapeutic

Figure 2. exoSTING selectively activates APC in the TME. exoSTING mediated delivery of CDNs drives selective induction of IFNβ by macrophages and dendritic cells.

A

0

1 × 1 0 4

2 × 1 0 4

3 × 1 0 4

4 × 1 0 4

5 × 1 0 4

1 0 - 6 1 0 - 5 1 0 - 4 1 0 - 3 1 0 - 2 1 0 - 1 1 0 0 1 0 1 1 0 2

S T I N G a g o n i s t ( µ M )

CD

86

(M

FI)

00

1 × 1 0 4

2 × 1 0 4

3 × 1 0 4

4 × 1 0 4

5 × 1 0 4

1 0 - 6 1 0 - 5 1 0 - 4 1 0 - 3 1 0 - 2 1 0 - 1 1 0 0 1 0 1 1 0 2

S T I N G a g o n i s t ( µ M )

CD

86

(M

FI)

0

Figure 3. exoSTING provides 100-1000 fold improvement in the potency of STING activation in human PBMC. IFNβ production following overnight treatment with exoSTING or free STING agonist (A). Activation of human DC (B) and monocytes (C) after exposure to exoSTING or free STING agonist as measured by CD86. The mean EC50 of IFNβ production (D) and maximal activation of mDCs (E) or monocytes (F) from 10 donors.

f r e e a g o n i s t e x o S T I N G0 . 0 1

0 . 1

1

1 0

1 0 0

co

nc

en

tra

tio

n

(µM

)

f r e e a g o n i s t e x o S T I N G0

2 × 1 0 4

4 × 1 0 4

6 × 1 0 4

8 × 1 0 4

1 × 1 0 5

CD

86

MF

I

f r e e a g o n i s t e x o S T I N G0

1 × 1 0 4

2 × 1 0 4

3 × 1 0 4

CD

86

MF

I

1 × 1 0 3

1 × 1 0 4

1 × 1 0 5

1 × 1 0 6

1 0 - 6 1 0 - 5 1 0 - 4 1 0 - 3 1 0 - 2 1 0 - 1 1 0 0 1 0 1 1 0 2

S T I N G a g o n i s t ( µ M )

IFN

β (

RLU

)

0

B C

D E FPBMC EC50 for IFNβ monocyte activationmDC activation

human PBMC monocytesmDC

Enhanced potency and selective DC activation

PBS 0.12 µg agonist 20 µg agonist exoSTING (0.0012 µg) exoSTING (0.012 µg) exoSTING (0.12 µg)

5 6 7 8 9 1 0 1 1 1 2 1 3 1 4 1 5 1 6 1 70

5 0 0

1 0 0 0

1 5 0 0

2 0 0 0

tum

or

vo

lum

e (

mm

3)

5 6 7 8 9 1 0 1 1 1 2 1 3 1 4 1 5 1 6 1 70

5 0 0

1 0 0 0

1 5 0 0

2 0 0 0

tum

or

vo

lum

e (

mm

3)

5 6 7 8 9 1 0 1 1 1 2 1 3 1 4 1 5 1 6 1 70

5 0 0

1 0 0 0

1 5 0 0

2 0 0 0

tum

or

vo

lum

e (

mm

3)

5 6 7 8 9 1 0 1 1 1 2 1 3 1 4 1 5 1 6 1 70

5 0 0

1 0 0 0

1 5 0 0

2 0 0 0

tum

or

vo

lum

e (

mm

3)

5 6 7 8 9 1 0 1 1 1 2 1 3 1 4 1 5 1 6 1 70

5 0 0

1 0 0 0

1 5 0 0

2 0 0 0

tum

or

vo

lum

e (

mm

3)

5 6 7 8 9 1 0 1 1 1 2 1 3 1 4 1 5 1 6 1 70

5 0 0

1 0 0 0

1 5 0 0

2 0 0 0

tum

or

vo

lum

e (

mm

3)

A

Potent anti-tumor activity in checkpoint-refractory tumors

C

5 6 7 8 9 1 0 1 1 1 2 1 3 1 4 1 5 1 6 1 70

5 0 0

1 0 0 0

1 5 0 0

2 0 0 0

d a y s p o s t i n o c u l a t i o n

tum

or

vo

lum

e (

mm

3)

B

P B S

free a

g o n is t (2

0µg )

free a

g o n is t (0

.12µ

g )

e x o S T ING

(0.0

0 1 2µg )

e x o S T ING

(0.0

1 2µg )

e x o S T ING

(0.1

2µg )

0

1 0

2 0

3 0

lun

g m

eta

sta

se

s

*E

PBS 0.12 µg agonist20 µg agonist

exoSTING (0.0012 µg) exoSTING (0.012 µg) exoSTING (0.120 µg)

D

Figure 4. exoSTING is more potent in inhibiting tumor growth than free STING agonist. Schematic for tumor inoculation of subcutaneous (SC) and intravenously (IV) inoculated tumors of the B16-F10 model (A). Mean tumor volumes for SC tumors, with treatment times indicated by yellow arrows (B). Individual spider plots of the SC tumor for each treatment arm (C). D, Representative images of excised lungs on day 17. Quantified lung metastases for each treatment arm (E). *, p<0.05 determined by one-way ANOVA.

P B S

2 0 ug a

g o n is t

0 .2 u

g ag o n is t

e x o S T ING

(0.2

ug )

0

5 0

1 0 0

1 5 0

IFNγ

sp

ots

/ 1

x1

05 c

ells *

5 6 7 8 9 1 0 1 1 1 2 1 3 1 4 1 5 1 6 1 70

5 0 0

1 0 0 0

1 5 0 0

d a y s p o s t i n o c u l a t i o n

tum

or

vo

lum

e (

mm

3)

A B

C

exoSTING induces systemic anti-tumor immunity

Figure 5. exoSTING drives systemic anti-tumor CD8 T cell responses that is enhanced by checkpoint blockade. Primary and secondary tumor growth in the B16-F10 model (A). PD1 blockade enhances the efficacy of a sub-optimal dose of exoSTING (B). Systemic antigen specific T cell response to B16-F10 epitopes 24 hours after the second dose in spleen (C). Mice were inoculated with B16-F10 and treated with exoSTING as above, with the indicated antibody administered 1 day prior to exoSTING treatment (D).

P r X- /

- ex o

S T I NG

n at i v

e e

x oS T I N

G

P r X e

x oS T I N

G

0

5 0

1 0 0

1 5 0

2 0 0

2 5 0

Sig

na

l/N

ois

e

*

A B

Figure 7. Protein X is required for optimal IFNβ production and enhanced efficacy of exoSTING. Representative dose-response of exoSTING stimulated human PBMCs with exosomes engineered to have enhanced expression of protein X (PrX), native expression of protein X, or be devoid of protein X (PrX-/-) (A). Maximum IFNβ production was diminished in PrX-/- exoSTING versus PrX exoSTING, *p<0.05 by one-way ANOVA (B). Efficacy of exoSTING PrX variants in the B16-F10 model, treated as in Figure 5, with a minimally efficacious does of exoSTING (0.02 µg of free agonist) (C).

0

5×1 0 4

1×1 0 5

2×1 0 5

2×1 0 5

1 0 - 61 0 - 51 0 - 41 0 - 31 0 - 21 0 - 1 1 0 0 1 0 1 1 0 2

S T IN G a g o n is t (µ M )

IFNβ

(R

LU)

0 6 9 1 2 1 5 1 8 2 10

5 0 0

1 0 0 0

1 5 0 0

2 0 0 0

2 5 0 0

d a y s p o s t i n o c u l a t i o n

Tum

or

Vo

lum

e (

mm

3)

C

Protein X is an exosome surface glycoprotein required for optimal potency of exoSTING

exoSTING induces minimal cell death in the tumor microenvironment

A

B

PBS

2 0 µg fr

e e ag o n is t

0 .2 µ

g fre e a

g o n is t

e x o S T ING

(0.2

µ g )0

5

1 0

1 5

2 0

2 5****

A B

Figure 9. exoSTING is selectively taken up by myeloid cells in injected tumors and does not kill tumor infiltrating leukocytes (TIL). Flow cytometry analysis of B16-F10 tumors one hour after IT injection of exoSTING labelled with Alexa Fluor 488 (AF488) (A). Analysis of TIL subsets by flow cytometry 24 hours after a second dose of free CDN or exoSTING, with dosing as in Figure 5 (B).

P B S

2 0 µg fr

e e ag o n is t

0 .2 µ

g fre e a

g o n is t

e x o S T ING

(0.2

µ g )0

5

1 0

1 5

2 0

2 5****

P B S

2 0 µg fr

e e ag o n is t

0 .2 µ

g fre e a

g o n is t

e x o S T ING

(0.2

µ g )0 .0

0 .5

1 .0

1 .5

2 .0

2 .5

C D 8 + T c

e lls

ma c ro

p h a g e s

d e n d r itic

ce lls

0

2 0

4 0

6 0

8 0

1 0 0

% o

f liv

e C

D45+

cel

ls

Selective delivery of exoSTING preserves the viability of tumor resident T-cells and APCs

• exoSTING was developed by exosome engineering technology (EngEXTM) to deliver a synthetic STING agonist that drives potent and systemic anti-tumor immunity.

• exoSTING demonstrates superior potency (100-1000 fold) through selective activation of APCs in vitro.

• The exosome surface glycoprotein protein X is required for optimal activity of exoSTING• exoSTING drives systemic CD8+ T cell anti-tumor responses in a model refractory to

checkpoint inhibition. • exoSTING blocked both primary and metastatic tumor growth at very low dose levels

(0.012 µg of agonist) and limits systemic cytokine release.• exoSTING induces selective STING agonism in APC of the TME, affording a potent anti-

tumor immune response with minimal collateral damage.

Summary

D

5 1 0 1 5 2 00

2 5 0

5 0 0

7 5 0

1 0 0 0

1 2 5 0

1 5 0 0

1 7 5 0

2 0 0 0

2 2 5 0

d a y s p o s t i n o c u l a t i o n

tum

or

vo

lum

e (

mm

3)

2 0 2 5 3 0 3 5 4 00

5 0 0

1 0 0 0

1 5 0 0

2 0 0 0

2 5 0 0

d a y s p o s t i n o c u l a t i o n

tum

or

vo

lum

e (

mm

3)

PBS 0.2 µg free agonist20 µg free agonist exoSTING (0.2 µg agonist)

2 µg free agonist 0.2 µg free agonist exoSTING (0.2 µg) PrX exoSTING (0.2 µg)

IFNβ

DAPI

FTM

pT

BK1

CD1

9 DA

PIFT

M

CC

3 DA

PIFT

M

4 HR

24 H

R

Quantitation

CIVO Microinjection Image Analysis

Figure 8. exoSTING is selective in targeting immune cells for IFNβ induction. Schematic for the Comparative In Vivo Oncology (CIVO®) Platform by Presage Biosciences (A). IFNβ RNA, pTBK1 IHC and CC3 IHC in the A20 lymphoma model show that exoSTING targets IFNβ induction of the STING pathway to immune cells (B). Massive cell death is observed in high dose CDN, corresponding to the efficacious dose in the B16F10 tumor model (each panel is a different injection site on the same tumor).

free agonist exoSTING free agonist exoSTING free agonist exoSTING

B16-F10 SC1x106 cells

B16-F10 IV1x105 cells

lungexcision

D0 D4 D5 D8 D11 D17

intra-tumoraldosing of SC tumor

4 HR 24 HR

3

2

1

tota

l IFN

βsig

nal

4 HR

0.8

0.6

0.4

0.2

24 HR

Frac

tion

CC

3+

IFNβ

CC3

exoSTING (0.2 µg)2 µg free agonist 0.2 µg free agonist

PrX exoSTING (0.2 µg)

60% CRs exoSTING60% CRs free agonist

100% protection exoSTING80% protection free agonist

B16-F10 primary challenge B16-F10 secondary challenge exoSTING combination with αPD1

PBS IgG + exoSTING(0.03 µg agonist)αPD1 αPD1 + exoSTING

(0.03 µg agonist)

B16-F10 CD8 depletion

7 8 9 1 0 1 1 1 2 1 3 1 4 1 5 1 6 1 70

3 0 0

6 0 0

9 0 0

1 2 0 0

1 5 0 0

1 8 0 0

d a y s p o s t i n o c u l a t i o n

tum

or

vo

lum

e (

mm

3)

PBS

IgG + exoSTINGαCD8

αCD8 + exoSTING

splenic CD8+ T cell response after 2nd IT dose

maximum IFNβ productionPBMC IFNβ production exoSTING PrX variants (0.02 µg)

free agonistPrX-/- exoSTINGnative exoSTINGPrX exoSTING

PBSPrX-/- exoSTING

PrX exoSTINGnative exoSTING

CIVO

tumor

CD8+ T cells macrophages dendritic cells1 hour B16-F10 TIL uptake

B16-F10 SC tumor

Presented at the 33rd Annual Meeting of the Society for Immuno-Therapy of Cancer, November 7-11th, 2018 in Washington, DC USA. All inquiries can be directed to presenting authors or by visiting www.codiakbio.com.

Exosomes carryingnatural STING agonists

DNAdamage

Exosome mediated immune activation

PD-1

PDL-1

Exosome mediated immunosuppression

Cancer Cell

B cellT cell

Dendritic cell

MacrophageNK cell

Cancercells

PrX

CDN

CDN release in APC

%A

F488

+of

cel

l sub

set

A Btumor RNA

PBS

a g o n is t (2

0 µg )

a g o n is t (0

.12µ

g )

e x o S T ING

(0.1

2 µg )

0

5 0 0

1 0 0 0

1 5 0 0

PB S

a g o n is t (2

0 µg )

a g o n is t (0

.12µ

g )

e x o S T ING

(0.1

2 µg )

0

2 0

4 0

6 0

8 0

1 0 0 *

serum cytokines

PB S

A g o n is t (2

0 µg )

A g o n is t (0

.12µ

g )

e x o S T ING

(0.1

2 µ g )0

2 0 0

4 0 0

6 0 0

8 0 0 ****

P B S

A g o n is t (2

0 µg )

A g o n is t (0

.12µ

g )

e x o S T ING

(0.1

2 µ g )0

2

4

6

8

1 0 **

exoSTING elicits minimal systemic cytokine responses

Figure 6. Cytokine responses following IT exoSTING administration are limited to the tumor. Mice bearing subcutaneous 50-70 mm3 B16-F10 tumors received IT injections of exoSTING or the indicated amount of free STING agonist. 4 hours after injection, tumors (A) and serum (B) were harvested and assayed for the indicated cytokines. *, p<0.05, **, p<0.005, ****, p<0.0005 by one- way ANOVA.

P B S

A g o n is t (2

0 µg )

A g o n is t (0

.12µ

g )

e x o S T ING

(0.1

2 µ g )0

1 0

2 0

3 0 *

IFNβ

(ng/

mL)

TNFα

(ng/

mL)

IL-6

(ng/

mL)

IFNβ TNFα IL-6

rela

tive

RNA

expr

essi

on

IFNβ CXCL10