Selective Activation of Antigen Presenting Cells by exoSTING Enhances

Tumor Antigen-Specific Immune Response

Su Chul Jang, Kelvin Zhang, Nuruddeen Lewis, Tong Zi, Joanne Lim, Rane A. Harrison, Raymond J. Moniz, Katherine Kirwin, Chang Ling Sia, Christine McCoy, Kevin

Dooley, Ke Xu, Jorge Sanchez-Salazar, Raymond W. Bourdeau, Agata Villiger-Oberbek, William K. Dahlberg, Shil Patel, Kyriakos D. Economides, Sriram

Sathyanarayanan

Codiak BioSciences, Cambridge, MA

Sample ID % Viable tumor % tumor cell Age Sex Tissue type Diagnosis

HC-17 90 70 72 Male Tongue SCC

HC-19 100 70 60 Female Oral Cavity SCC

HC-22 80 50 38 Male Tongue SCC

HC-24 85 60 59 Male Oral Cavity SCC

6 7 8 9 10 11 12 13 14 15 160

500

1000

1500

Injected tumors

Days

Tu

mo

r Vo

lum

e (m

m3)

PBS

Exosomes

FSA (100 g)

exoSTING (0.2 g)

789101112131415160

500

1000

1500

Contralateral tumors

Days

Tu

mo

r V

olu

me (

mm

3)

********

Exo

so

me

s

4h

24

h

4h

24

h

4h

24

h

4h

24

h

0

200

400

600

CD8+ Cells

CD

8 C

ells/m

m2

1 dose 2 doses 1 dose 2 doses

FSA (20 g) exoSTING (0.1 g)

* *

4-fold increase

vs. exosomes

2-fold decrease

vs. exosomes

Exo

so

me

s

4h

24

h

4h

24

h

4h

24

h

4h

24

h

0

5

10

15

IFN

% p

osit

ive a

rea o

f IF

N

1 dose 2 doses 1 dose 2 doses

FSA (20 g) exoSTING (0.1 g)

* n.s.

Exo

so

me

s

4h

24

h

4h

24

h

4h

24

h

4h

24

h

0

500

1000

1500

2000

F4/80+ Cells

F4/8

0 C

ells/m

m2

1 dose 2 doses 1 dose 2 doses

FSA (20 g) exoSTING (0.1 g)

Presented at the 34th Annual Meeting of the Society for Immuno-Therapy of Cancer, November 6-10th, 2019 in National Harbor, MD USA.

PBS FSA

(0.1 μg)exoSTING

(0.1 μg)

17 19 22 24 17 19 22 24 17 19 22 24Patient #

PBS

FSA (0.1

g)

exoS

TING (0

.1

g)0

5

10

15

IFN

Lo

g2 n

orm

alized

exp

ressio

n 1719

22

24

PBS

FSA (0.1

g)

exoS

TING (0

.1

g)4

6

8

10

12

14

CXCL9

Lo

g2 n

orm

alized

exp

ressio

n 1719

22

24

PBS

FSA (0.1

g)

exoS

TING (0

.1

g)4

6

8

10

12

14

CXCL10

Lo

g2 n

orm

alized

exp

ressio

n 1719

22

24

PBS

FSA (0.1

g)

exoS

TING (0

.1

g)4

5

6

7

8

IFN

Lo

g2 n

orm

alized

exp

ressio

n 1719

22

24

PBS

FSA (0.1

g)

exoS

TING (0

.1

g)7

8

9

10

CCL3

Lo

g2 n

orm

alized

exp

ressio

n 1719

22

24

PBS

FSA (0.1

g)

exoS

TING (0

.1

g)7

8

9

10

11

CCL3L1

Lo

g2 n

orm

alized

exp

ressio

n 1719

22

24

PBS

FSA (0.1

g)

exoS

TING (0

.1

g)7

8

9

10

11

CCL4

Lo

g2 n

orm

alized

exp

ressio

n 1719

22

24

-50 0 50 100 150 200

0.0

0.2

0.4

0.6

0.8

exoSTING (0.1 g)

Time (Hrs)

To

tal L

esio

n G

lyc

oly

sis

(g

)

Contralateral tumor

Injected tumor

-50 0 50 100 150 200

0.0

0.2

0.4

0.6

0.8

FSA (20 g)

Time (Hrs)

To

tal L

esio

n G

lyc

oly

sis

(g

)

Contralateral tumor

Injected tumor

Histology

Tumor

Acquisition

Fragments

(~5 mm3)Embed into Agarose

and InjectHisto-culture for

Four Hours

Gene Expression

Analysis

D0 D11

B16F10, SC

1E6/mouse

D8 D9 D124 h4 h

IHC IHC

A-E, C57BL/6 mice were implanted subcutaneously with 1×106 B16F10 cell on the right flank of

mice. FSA (20 μg) and exoSTING (0.1 μg) were injected intratumorally at Day 1 and Day 4 (n = 5

per group). Tumors were collected at 4 and 24 hours after the first dose and second doses. Tumor

sections were stained with H&E, IFNβ mRNA, CD8, and F4/80 (B). IFNβ positive area (C), CD8

positive cells (D), and F4/80 positive cells (E) were measured. CD8 cells were 4-fold higher in

exoSTING treated tumors than an exosome control. *, P < 0.05 by two-way ANOVA with Tukey’s

multiple comparison test, compared to exosome control. n.s.; non-significant

A-D. Four hours after intra-tumoral injection of PBS, FSA (0.1, 20, and 100 μg), and exoSTING

(0.001, 0.01, and 0.1 μg) into B16F10 tumors (n = 4 per group), differentially expressed genes

were analyzed using NanoString technology. Relative expression of IFNβ (A), CXCL9 (B), and

IFNγ (C). D, Heatmap of differentially expressed genes between FSA and exoSTING. Blue box;

genes that are down-regulated by exoSTING more than FSA, Red box; genes that are up-

regulated by exoSTING more than FSA, Green box; genes that are down-regulated by both

exoSTING and FSA.

A, Fresh human tumor tissues were sliced into 5 x 5 mm size and injected with PBS, FSA (0.1 μg)

or exoSTING (0.1 μg) intratumorally. After 4 hours treatment, RNA was isolated and analyzed by

NanoString. B, Patient information and basic characterization of tumor tissue after H&E staining.

C, The heatmap of top 20 up-regulated genes from NanoString analysis. D, Gene Set Enrichment

Analysis of up- and down-regulated pathway in exoSTING (0.1 μg) treated tumors, compared to

FSA (0.1 μg) treated tumors. E, Relative expression of IFNβ, CXCL9, CXCL10, IFNγ, CCL3,

CCL4, and CCL3L1, measured by NanoString.

A, B16F10 tumor cells were implanted subcutaneously in the right (1×106 cells) and left (5×105

cells) flanks of mice (n = 10 per group). When tumor volumes reached 50 – 100 mm3, testing

agents were injected intratumorally into right flank tumors 3 times at 3 days interval. Red arrows in

the graph indicate intra-tumoral injection days. Both injected and non-injected contralateral tumor

growth was measured. ****, P < 0.0001 by two-way ANOVA with Tukey’s multiple comparison test,

compared to both PBS and exosomes. B-E, When tumor volumes reached 50 – 100 mm3, testing

agents were injected intratumorally into right flank tumors twice with 3 days interval. For PET-CT,18F-FDG was injected intravenously at day -1, 1, 2, 3, and 6. Total lesion glycolysis was measured

in both injected and non-injected contralateral tumors after FSA (20 μg) (B) and exoSTING (0.1

μg) (C) injection (n = 3-8 per group). Representative PET-CT images of FSA (20 μg) (D) and

exoSTING (0.1 μg) (E) groups. Cont.; contralateral tumor, Inj.; injected tumor

•ExoSTING was preferentially taken up by differentiated APCs. In vitro and in vivo studies

showed more than a 100-fold increase in potency by exoSTING treatment compared to

FSA.

•Intra-tumoral administration of exoSTING in B16F10 tumors preserves the viability of T

cells and APCs, reduces collateral tissue damage, and eventually recruits more T cells into

tumors, whereas FSA induces immune cell ablation.

•ExoSTING produced a systemic tumor-specific T cell response as demonstrated by growth

inhibition of non-injected distal tumors and produced memory responses against tumors.

•ExoSTING activated interferon stimulated genes, including IFNβ, CXCL9, CXCL10, and

IFNγ in ex vivo human tumor histo-culture, more than equivalent amounts of FSA.

•Clinical development for exoSTING is planned to begin in the first half of 2020.

A

B

C D

E

A

B C

D E

Inj.Con.Inj.

Con.Inj.

Con.

FSA (20 μg)

Inj.Con.

Inj.Con.Inj.Con.

exoSTING (0.1 μg)

A

B

C D E

A B C

D

B16F10 tumor cells were implanted subcutaneously in the right (1×106 cells) flank of mice (n = 5-

10 per group). When tumor volumes reached 50 – 100 mm3, testing agents were injected

intratumorally into right flank tumors. Red arrows in the graph indicate intra-tumoral injection days.

B16F10 cells (1×106 cells) were implanted to mice that had CR and naïve mice (n = 5) on day 20

to the left flank. Primary (A) and re-challenged (B) tumor growth were measured.

exoSTING Increases T Cell Infiltration in Tumor

Microenvironment Without Immune Cell Ablation

exoSTING Preserves Memory Responses Against Tumors

exoSTING Activates STING Pathway in ex vivo Human Tumor

Tissues After Intra-Tumoral Administration

exoSTING Inhibits Tumor Growth in Both Injected and Non-

Injected Contralateral Tumors

Summary

exoSTING Selectively Activates M2 Immunosuppressive

Macrophages

exoSTING Induces Immune Activation Pathways in Tumor

Without Bell-Shaped Dose Response

FSA (0.1 μg) vs. exoSTING (0.1 μg)

Introduction

•Exosomes have shown potential to carry STING agonists to antigen presenting cells

(APCs), resulting in an anti-tumor immune response.

•ExoSTING™ is composed of engineered exosomes overexpressing PTGFRN and loaded

with a cyclic dinucleotide (CDN) small molecule STING agonist to leverage the inherent

ability of exosomes to deliver STING agonists selectively to APCs.

•ExoSTING selectively activates the STING pathway in tumor-resident APCs, demonstrating

greater potency than free STING agonist (FSA) and without immune ablation.

Various concentrations of FSA and exoSTING were incubated with M2 (A) or M1 (B) polarized

human macrophages, or stimulated T cells (C), and production of IFNβ in supernatant was

measured. Monocytes were purified from human peripheral blood mononuclear cell (PBMCs) and

differentiated into M1 or M2 macrophages by IFNγ or combination of IL-4, TGFβ, and IL-10,

respectively. T cells were purified from human PBMCs and stimulated with anti-CD3/anti-CD28.

4

6

8

10

12

IFN

Lo

g2 n

orm

alized

exp

ressio

n

0.001 0.01 0.1 0.1 20 100

exoSTING (g) FSA (g)

PBS

8

9

10

11

12

13

CXCL9

Lo

g2 n

orm

alized

exp

ressio

n

0.001 0.01 0.1 0.1 20 100PBS

exoSTING (g) FSA (g)

4.0

4.5

5.0

5.5

6.0

6.5

IFN

Lo

g2 n

orm

alized

exp

ressio

n

0.001 0.01 0.1 0.1 20 100PBS

exoSTING (g) FSA (g)

0

50000

100000

150000

10 -6 10 -5 10 -4 10 -3 10 -2 10 -1 100 101 102

M2 Polarized Macrophage

STING agonist (M)

IFN

(R

LU

)

0

FSA

exoSTING

EC50=0.05 M

EC50=2.4 M

0

50000

100000

150000

200000

10 -6 10 -5 10 -4 10 -3 10 -2 10 -1 100 101 102

M1 Polarized Macrophage

STING agonist (M)

IFN

(R

LU

)

0

FSA

exoSTING

EC50=20.6 M

0

1000

2000

3000

10 -6 10 -5 10 -4 10 -3 10 -2 10 -1 100 101 102

Stimulated T Cells

STING agonist (M)

IFN

(R

LU

)

0

FSA

exoSTING

EC50=3.7 M

A B C

exoSTING (0.1 μg)FSA (20 μg)

Dose 1-4 h Dose 1-24 h Dose 2-4 h Dose 2-24 h

H&

EC

D8

F4

/80

IFNβ

Dose 1-4 h Dose 1-24 h Dose 2-4 h Dose 2-24 h

PBS

FSA (μg)

0.001 0.01 0.1 0.1 20 100

exoSTING (μg)

6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 220

1000

2000

3000

Primary tumor

Days

Tu

mo

r V

olu

me (

mm

3)

PBS

Exosomes

FSA (100 g)

exoSTING (0.2 g)

CR 10/10

CR 4/10

5 10 15 20 25 30 35 400

500

1000

1500

2000

2500

Re-challenged tumor

Days

Tu

mo

r V

olu

me (

mm

3)

Naive

FSA (100 g)

exoSTING (0.2 g)

A B