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444: ENGINEERED EXOSOMES EFFICIENTLY DELIVER STAT6 ANTISENSE OLIGONUCLEOTIDES TO TUMOR ASSOCIATED MACROPHAGES (TAMS) RESULTING IN POTENT LOCAL AND SYSTEMIC ANTI-TUMOR ACTIVITYSushrut Kamerkar1, Charan Leng1, Olga Burenkova1, Su Chul Jang1,Christine McCoy1, Tong Zi1, Kelvin Zhang1, William Dahlberg1, Kyriakos Economides1, Timothy Soos1, Dalia Burzyn1, Sriram Sathyanarayanan1

1Codiak BioSciences, Cambridge, MA

Session: Oligonucleotide TherapeuticsAbstract Number: 444

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Introduction

2

Anti-tumoralF

att

y A

cid

Pro-tumoral

LP

S

Tumor associated macrophages (TAMs) are potent drivers of an

immunosuppressive tumor microenvironment

Experimental therapies targeting TAM differentiation, recruitment or survival

have shown marginal anti tumor efficacy

Alternative approach→ reprogramming from an immunosuppressive M2 to an

anti-tumoral pro-inflammatory M1 phenotype

Targeting key TAM transcription factor STAT6 could

effectively reprogram M2 macrophages to anti-tumoral M1 macrophages

Codiak has developed novel, engineered exosomes that

selectively deliver antisense oligonucleotides (ASO) targeting STAT6, to M2 macrophages

exoASO™

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Exosomes mediate preferential delivery of ASOs to myeloid cells and TAMs in vivo

3

B c

ells

T cel

ls

NK c

ells

Monocy

tes

gMDSC/n

eutrophil

mM

DSCs

CD11

b+ DCs

CD11

b- DCs

0

50000

100000

150000

200000

Cy

5 M

FI (

min

us

PB

S c

on

tro

l)

Exo ASO

Free ASO

✱✱✱✱

✱✱✱✱✱✱✱✱

11x

11x12x

B c

ell

T cel

l

NK c

ell

Mac

s

Red

pulp

mac

s

Monocy

tes

mM

DSC

gMDSCs/

neutr

0

10000

20000

30000

40000

50000

Cy

5 M

FI (

min

us

PB

S c

on

tro

l)

Exo ASO

Free ASO

✱✱✱✱

✱✱✱✱✱✱✱✱

3.5x 2.2x2.7x

CD45

-

CD11

b+

Mac

rophag

es

CD11

b-

Mac

rophag

es B c

ells

T cel

ls

0

5000

10000

15000

2000040000

50000

60000

Cy

5 M

FI (

min

us

PB

S c

on

tro

l)

Exo ASOFree ASO

✱✱

6x

✱✱✱✱

12x

CD45

-

Kupffe

r ce

lls

0

500000

1000000

1500000

2000000

Cy

5 M

FI (

min

us

P

BS

co

ntr

ol)

Exo ASO

Free ASO✱

✱✱✱

9x

CD45

-

Mac

rophag

es

NK c

ells

T cel

lsDCs

B c

ells

mM

DSCs

gMDSCs

0

10000

20000

30000

40000

Cy

5 M

FI (

min

us

P

BS

co

ntr

ol) Exo ASO

Free ASO

✱

✱✱✱

✱✱✱✱

1.3x

3.5x

5x

BLOOD (IV) LIVER (IV) SPLEEN (IV)

BONE MARROW (IV) TUMOR (IV)

A B C

D E

Exosome tropism to myeloid cells promotes selective delivery of

ASOs. A-E: BALB/c mice bearing CT26 subcutaneous (SC) tumors

received one intravenous (IV) dose (8μg) of fluorescently-labeled Cy5

exoASO or free ASO. One hour later, peripheral blood (A), liver (B),

spleen (C), bone marrow (D) and tumor (E) were dissected and

enzymatically digested, and cell suspensions analyzed by flow cytometry.

****, P < 0.0001, ***, P < 0.001, **, P < 0.01 and *, P < 0.05 by two-way

ANOVA with Sidak’s multiple comparison test.

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Effective and durable STAT6 silencing in TAMs by exoASO-STAT6

4

0 2 4 6 8 10 12 14 16

0

20

40

60

80

100

120

140

160

Timepoint (Days)

No

rmalize

d %

Pro

tein

exp

ress

ion

(h

ST

AT

6)

1Day 10

0

20

40

60

80

100

120

102 103 104

concentration (nM)

No

rmalized

% g

en

e e

xp

ressio

n (h

ST

AT

6)

0

4 days

7 days

0 2 4 6 8 10 12

0

20

40

60

80

100

120

140

Timepoint (Days)

No

rmalized

% P

rote

in e

xp

ressio

n (m

ST

AT

6)

0 2 4 6 8 10 12 14 16

0

20

40

60

80

100

120

140

160

Timepoint (Days)

No

rmalize

d %

Pro

tein

exp

ress

ion

(h

ST

AT

6)

exoASO-STAT6 2.5 uM

STAT6 Free ASO 2.5 uM

exoASO-Scramble 2.5 uM

Day 7

A B CHuman M2 Macrophages Mouse M2 Macrophages Cyno M2 Macrophages

2 3 4 1 2 3 4 1 3 4

Potent and durable knockdown of STAT6 across species. Human monocyte derived M2 macrophages (A), mouse bone marrow derived M2 macrophages (B),

and cynomolgus monkey monocyte derived M2 macrophages (C) were incubated for 24h with equivalent doses of exoASO STAT6 and free STAT6 ASO, along with

an exoASO Scramble control. Expression of STAT6 protein was quantified over time by automated western blot (WES™). Legend: (1: exoASO STAT6, 2: Free

STAT6 ASO, 3: exoASO Scramble, 4: Untreated)

Day 3

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Potent monotherapy activity by exoASO-STAT6 treatment in CT26 tumors

5

8 10 12 14 16 18 20 22 24 26 28

0

500

1000

1500

2000

2500

Days post re-challenge(Mice rechallenged 42 days post first tumor inoculation)

Tu

mo

r V

olu

me, m

m3

Control (Naive)

exoASO STAT6 TIW (2wk) 6ug (n=7)

exoASO STAT6 BIW (3wk) 6ug (n=8)

A B C

8 12 16 20 24

0

400

800

1200

1600

Days Post Tumor Implantation

Tu

mo

r V

olu

me, m

m3

PBS TIW (2wk)

exoASO Scramble TIW (2wk) 6ug

exoASO STAT6 TIW (2wk) 6ug

exoASO STAT6 BIW (3wk) 6ug

10 20 30 40

0

500

1000

1500

2000

2500

Days Post Tumor Implantation

Tu

mo

r V

olu

me, m

m3

10 20 30 40

0

500

1000

1500

2000

2500

Days Post Tumor Implantation

Tu

mo

r V

olu

me, m

m3

PBS

exoASO Scramble

exoASO STAT6 18 ug

exoASO STAT6 6 ug

exoASO STAT6 2 ug

exoASO STAT6 0.67 ug

exoASO STAT6 0.22 ug

Exo Only

Monotherapy efficacy in CT26 is dependent on cumulative dose and induces immunological memory. CT26 tumor cells were implanted SC in the flanks of

mice (n=10 per group). A: Tumor growth volumes of mice injected intratumorally with PBS, exosomes only, exoASO Scramble (6ug) (BIW, 3wk) or exoASO STAT6

(0.22, 0.67, 2, 6 and 18ug) (BIW, 3wk), n=10 mice per group. B: Tumor growth volumes of mice injected intratumorally with PBS (TIW, 2wk), exoASO STAT6 (6ug)

(BIW, 3wk) or (TIW, 2wk) or exoASO Scramble (6ug) (TIW, 2wk). C: Tumor growth volumes of mice re-challenged with CT26 tumors on the opposite flank of

complete responders from B, naïve Balb/C mice bearing SC CT26 tumors were used as controls. (BIW: 2x a week, TIW: 3x a week)

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exoASO-STAT6 monotherapy efficacy in CT26 tumors is CD8 T-cell dependent

6

5 10 15 20 25

0

1000

2000

3000

Days Post Tumor Implantation

Tu

mo

r V

olu

me, m

m3

-0.05

0.00

0.05

0.10

0.15

Tu

mo

r G

row

th R

ate

******

5 10 15 20 25

0

1000

2000

3000

Days Post Tumor Implantation

Tu

mo

r V

olu

me, m

m3

exoASO Scramble (6ug) + All Isotypes

exoASO Scramble (6ug) + antiCD8

exoASO Scramble (6ug) + antiCD4

exoASO STAT6 (6ug) + All Isotypes

exoASO STAT6 (6ug) + antiCD8

exoASO STAT6 (6ug) + antiCD4

A B

Monotherapy efficacy in CT26 is dependent on CD8 T-cells. CT26 tumor cells were implanted SC in the flanks of mice (n=10 per group). A-B: Tumor growth

volumes (A) and tumor growth rates (B) of after CD8 and CD4 T cell depletion, injected intratumorally with either exoASO Scramble (6ug) (TIW, 2wk) or exoASO

STAT6 (6ug) (TIW, 2wk). Mice received one dose of 10mg/kg of anti-CD8 or anti-CD4 antibody, or 10mg/kg of isotype control antibody, prior to IT injections, and

BIW thereafter. ****, P < 0.0001, **, P < 0.01 by one-way ANOVA with Tukey’s multiple comparison test. (BIW: 2x a week).

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exoASO-STAT6 monotherapy results in remodeling of the tumor microenvironment in CT26 tumors

7

exoASO STAT6exoASO Scramble

Retnla (Fizz-1) (M2)

Nos2 (M1)

exoASO STAT6exoASO Scramble

exoASO Scramble exoASO STAT6

Intra tumoral administration of exoASO STAT6 leads to remodeling of the tumor microenvironment

in CT26. CT26 tumor cells were implanted SC in the flanks of mice (n=3 mice per group). exoASO STAT6

(6ug) and exoASO Scramble (6ug) were dosed IT, (TIW, 1 week). A: tSNE plots from scRNA seq data, of

intra tumoral cells from exoASO Scramble or exoASO STAT6 group, to identify individual immune cell

populations. B: tSNE plots from data from (A), showing global changes in expression of Nos2 and Retnla

(Fizz-1) in immune cell populations

AB

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Systemic administration of exoASO-STAT6 results in monotherapy activity in HCC orthotopic model

8

D0

Inoculation

IV injection, TIWx2, 6 total

D3 D14 D16

IP injection, DIWx2, 4 total

Randomization

End of Study

EXOSOMES ONLY SCRAMBLE EXOASO

STAT6 EXOASO

Exo

som

es O

nly

exoA

SO S

cram

ble

exoA

SO S

TAT6

0

10

20

30

40

LW/BW ratio

% L

ive

r w

eig

ht/

Bo

dy w

eig

ht ****

****

Untr

eate

d

aCSF1R

aPD-1

0

10

20

30

40

LW/BW ratio

Exoso

mes

Only

exoA

SO S

cram

ble

exoA

SO

STA

T6

0

20

40

60

80

% Tumor cells

% T

um

or

cells

*******

Exosomes Only

exoASO Scramble

exoASO STAT6

A

B C

Anti-tumor monotherapy activity and reprogramming of the TME by exoASO STAT6 in HCC . Hepa1-6 cells were implanted in the livers of C57BL/6J by partial

hepactectomy, followed by splenectomy (n=8 mice per group). ExoASOs (12ug/dose) and exosomes only were administered by intravenous injections, while

antibodies were dosed intraperitoneally following dosing regimen in (A). A: Dosing scheme. B: Efficacy as determined by % of liver weight/body weight ratio, images

of resected livers at end point. C: Percentage (%) of tumor cells as calculated by Halo Imaging Analysis Platform from H&E-stained sections of livers ****, P <

0.0001, ***, P < 0.001, **, P < 0.01 and *, P < 0.05 by one-way ANOVA with Tukey’s multiple comparison test.

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Systemic administration of exoASO STAT6-in HCC tumor model results in iNOS induction and reprogramming of the TME

9

Exosomes Only

exoASO Scramble

exoASO STAT6

0.0

0.5

1.0

1.5

Stat6

No

rmalized

ST

AT

6 m

RN

A e

xp

ressio

n

********

-4 -2 0 2 4 6

0

1

2

3

4

5

6

7

8

Log 2 (Fold change exoASO STAT6 vs exoASO Scramble baseline)

-lo

g10

(P

valu

e) Myc Cd276

Ccl17

Mmp9

Cd3e

Cd8aCd8b1

Cd3gCd3dTgfb1

Cd86Cd80

p<0.05

Exosomes Only

exoASO Scramble

exoASO STAT6

0

50

100

150

200

250

Ccl17

mR

NA

no

rmalized

lin

ear

co

un

ts ****

*

Exosomes Only

exoASO Scramble

exoASO STAT6

0

500

1000

1500

2000

2500

Myc

mR

NA

no

rmalized

lin

ear

co

un

ts ********

Exosomes Only

exoASO Scramble

exoASO STAT6

0

50

100

150

200

Cd276

mR

NA

no

rmalized

lin

ear

co

un

ts *******

Exosomes Only

exoASO Scramble

exoASO STAT6

0

100

200

300

400

Icos

mR

NA

no

rmalized

lin

ear

co

un

ts

*****

Exosomes Only

exoASO Scramble

exoASO STAT6

-8

-6

-4

-2

0

2

4

Cell proliferation

Cell P

rolife

rati

on

Sco

re ********

Exosomes Only

exoASO Scramble

exoASO STAT6

-4

-3

-2

-1

0

1

2

TGF beta Signaling

TG

F b

eta

Sig

nalin

g S

co

re

********

Exosomes Only

exoASO Scramble

exoASO STAT6

0

50

100

150

200

250

CD8 T cell

CD

8 T

ce

ll S

co

re

***

Exosomes Only

exoASO Scramble

exoASO STAT6

0

100

200

300

400

500

Cytotoxic cells

Cyto

toxic

cells S

co

re

**

Exosomes Only

exoASO Scramble

exoASO STAT6

0.0

0.2

0.4

0.6

0.8

% FoxP3+ T cells

% F

oxP

3+

cells

**

Exosomes Only

exoASO Scramble

exoASO STAT6

0

5

10

15

20

% CD8+ T cells

% C

D8

+ c

ells

****

CD

8/D

AP

I

Exosomes Only exoASO Scramble exoASO STAT6

Fo

xP

3/D

AP

IS

TA

T6/D

AP

I

Exosomes Only

exoASO Scramble

exoASO STAT6

0

20

40

60

80

100

% Stat6 +ve cells

% S

tat6

+ c

ell

s

******

iNO

S

Exosomes Only

exoASO Scramble

exoASO STAT6

0

10

20

30

% iNOS+ in tumor

% N

os

2

**

DA

B C

Anti-tumor monotherapy activity and reprogramming of the TME by exoASO STAT6 in HCC A-B: Gene

expression analysis (A) and pathway and cell type scoring (B) by Nanostring using the nCounter Mouse PanCancer IO

360 panel from whole liver tissue. C: STAT6 knockdown in whole liver tissue at study end point. D: Expression of

STAT6, CD8, FoxP3 and iNOS by IHC.

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exoASO™: A novel engineered exosome therapeutic

10

• exoASO is a novel, engineered exosome that can

selectively deliver antisense oligonucleotides to

tumor associated M2 macrophages.

• exoASO enables selective silencing of STAT6, a key

transcription factor that controls the

immunosuppressive program.

• Effective silencing of STAT6 both in vitro and in vivo,

and across species, lead to the modulation of key

factors involved in the M2→M1 transition

• Intratumoral administration of exoASO-STAT6 resulted

in a potent monotherapy response that is dependent

on cumulative dose

• exoASO STAT6 treatment induced immunological

memory, and demonstrated an establishment of an

adaptive anti-tumor response mediated by cytotoxic

CD8 T cells

exoASO™

exoASOs against STAT6 represent a first-in-class strategy to target tumor-

associated myeloid cells in a highly selective manner

• Single cell RNA sequencing confirmed an

increase in Nos2+ macrophage population, and

a reduction in Retnla (Fizz1)+ macrophage

population

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