0 6 12 18 24 30 36 42 480

5×107

1×108

1.5×108

Time (Hours)

To

tal O

bje

ct

Inte

nsit

y (R

CU

x u

m/im

ag

e)

Exo-ASO-M2

Free-ASO-M2

Exo-ASO M0

Free ASO-M0

Exosome mediated delivery of antisense oligonucleotides reprograms tumor-

associated macrophages and induces anti-tumor responses

Presented at the 23rd Annual Meeting of the American Society of Gene & Cell Therapy, May 12-15th, 2020 in Boston, MA USA.

Sushrut Kamerkar1, Dalia Burzyn1, Charan Leng1, Olga Burenkova1, Su Chul Jang1, Raymond Yang1, Katherine Kirwin1, Tong Zi1, William Dahlberg1,

Eric Zhang1, Kelvin Zhang1, Joyoti Dey2, Marc Grenley2, Emily Beirne2, Scott Estes1, Kyriakos Economides1, Timothy Soos1, Sriram Sathyanarayanan1

1Codiak BioSciences, Cambridge, MA2Presage BioSciences, Seattle, WA

C/EBPβ mRNA silencing by exoASO effectively repolarizes

human M2 macrophages

Potent monotherapy and combination anti-tumor activity

with C/EBPβ exoASO

Robust induction of pro-inflammatory M1 markers in vivo

with C/EBPβ exoASO

Exosome mediated selective delivery of ASOs to TAMs in vivo

Summary

exoASO™: A novel engineered exosome

Cholesterol-tagged ASOs load efficiently on exosomes

Introduction

Effective silencing of C/EBPβ in vivo in TAMs by exoASO

Effective reprogramming of M2 macrophages in vivo by

knockdown of C/EBPβ

Potent target gene silencing in vitro by C/EBPβ exoASO

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 C/EBPβ

could effectively reprogram M2 macrophages

to anti-tumoral M1 macrophages

Codiak has developed novel, engineered

exosomes that selectively deliver antisense

oligonucleotides (ASO) targeting C/EBPβ, to M2

macrophages

EXOSOMES

• 30-200nm lipid/protein nanoparticle

• Natural inter-cellular

communication system for

macromolecules

• Off-the-shelf immune silent

drug delivery

exoASO

• exoASO is a novel engineered

exosome, stably loaded with

cholesterol tagged antisense

oligos• ~4000 copies/exosome

DELIVERY TO TAMs

• Selective targeting of M2

macrophages in the tumor microenvironment (TME)

TARGETING C/EBPβ

• Key transcriptional regulator of

M2 immune-suppressive macrophages

Post clean-up exosome pellets Uptake of Cy5 exoASO in macrophages

0.0

0.2

0.4

0.6

0.8

1.0

1.2

102 103 104

CEBP

concentration (nM)No

rma

lize

d %

ge

ne

ex

pre

ssio

n (h

CE

BP

B)

0

exoASO CEBPB

Free CEBPB ASO

Untreated

exoASO Scramble

0.0

0.2

0.4

0.6

0.8

1.0

1.2

102 103 104

CD163

concentration (nM)No

rma

lize

d %

ge

ne

ex

pre

ss

ion

(h

CD

16

3)

0

exoASO CEBPB

Free CEBPB ASO

Untreated

exoASO Scramble

Untr

eate

d

exoA

SO S

cram

ble

exoA

SO C

ebpB

Free

Ceb

pB A

SO

0

20

40

60

80

100

CEBP

mR

NA

no

rmalized

lin

ear

co

un

ts

********

Untr

eate

d

exoA

SO S

cram

ble

exoA

SO C

ebpB

Free

Ceb

pB A

SO

0

200

400

600

800

CD163

mR

NA

no

rmalized

lin

ear

co

un

ts

***

***

Untr

eate

d

exoA

SO S

cram

ble

exoA

SO C

ebpB

Free

Ceb

pB A

SO

0

100

200

300

CD206

mR

NA

no

rmalized

lin

ear

co

un

ts

******

Untr

eate

d

exoA

SO S

cram

ble

exoA

SO C

ebpB

Free

Ceb

pB A

SO

0

10

20

30

IL12b

mR

NA

no

rmalized

lin

ear

co

un

ts

*****

Untr

eate

d

exoA

SO S

cram

ble

exoA

SO C

ebpB

Free

Ceb

pB A

SO

0

1000

2000

3000

4000

IL10

IL10 p

g/m

l

Untr

eate

d

exoA

SO S

cram

ble

exoA

SO C

ebpB

Free

Ceb

pB A

SO

0

500

1000

1500

TNF

TN

Fa p

g/m

l

Untr

eate

d

exoA

SO S

cram

ble

exoA

SO C

ebpB

Free

Ceb

pB A

SO

0

10

20

30

40

50

TARC/CCL17

TA

RC

/CC

L17 p

g/m

l

Untr

eate

d

exoA

SO S

cram

ble

exoA

SO C

ebpB

Free

Ceb

pB A

SO

0

50

100

150

200

250

IL12p40

IL12p

40 p

g/m

l

GENE EXPRESSION ANALYSIS

CYTOKINE PRODUCTION

CD45

+

CD45

-

Kupff

er c

ells

CD11

b+ F4/

80-

CD11

b+ Ly6

C+

CD11

b+ Ly6

G+

0

500000

1000000

1500000

2000000

Cy

5 M

FI (

min

us

P

BS

co

ntr

ol)

Exo ASO

Free ASO

B c

ells

T cel

ls

NK c

ells

CD11

b+ DCs

CD11

b- DCs

Monocy

tes

mM

DSCs/

other

gMDSC/n

eutrophil

0

50000

100000

150000

200000

Cy

5 M

FI (

min

us

PB

S c

on

tro

l)

Exo ASO

Free ASO

LIVER (IV) BLOOD (IV)

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

SPLEEN (IV)

No

rma

lize

d c

ou

nts

No

rma

lize

d c

ou

nts

ASO-Cy5 ASO-Cy5

TUMOR (IT)

— Unlabeled exo

— Macrophages_exoASO

— Tumor cells_exoASO

— Unlabeled exo

— Macrophages_exoASO

— gMDSC_exoASO

— mMDSC_exoASO

— cDC2_exoASO

— cDC1_exoASO

exoASOScramble

exoASOCebpB

exoASOScramble

exoASOCebpB

0.00

0.25

0.50

0.75

1.00

1.25

1.50

No

rma

lize

d G

en

e E

xp

ressio

n (m

Ceb

pB

)

**

Non enriched CD11b Enriched

*

exoASOScramble

exoASOCebpB

exoASOScramble

exoASOCebpB

0.00

0.25

0.50

0.75

1.00

1.25

1.50

1.75

No

rmalized

Gen

e E

xp

ressio

n (m

Arg

1)

***

Non enriched CD11b Enriched

C/EBPβ ARG-1

exoASOScramble

exoASOCebpB

0

1000

2000

3000

CD206

mR

NA

no

rmalized

lin

ear

co

un

ts

exoASOScramble

exoASOCebpB

3000

3500

4000

4500

5000

5500

Arg 1

mR

NA

no

rmalized

lin

ear

co

un

ts

exoASOScramble

exoASOCebpB

0

50

100

150

200

250

iNOS

mR

NA

no

rmalized

lin

ear

co

un

ts

Exo Only

WT Exo

PTGFRNExo

0

1000

2000

3000

4000

5000

AS

Os

/ex

os

om

e

exoASOScramble

exoASOCebpB

0

20

40

60

IFN1

mR

NA

no

rmalized

lin

ear

co

un

ts

exoASOScramble

exoASOCebpB

0

500

1000

1500

TGF1

mR

NA

no

rmalized

lin

ear

co

un

ts

exoASOScramble

exoASOCebpB

0

500

1000

1500

2000

2500

CSF1r

mR

NA

no

rmalized

lin

ear

co

un

ts

exoASO

Scramble

exoASO

C/EBPβ

8 10 12 14 16 18 20 22 24 26 28 300

250

500

750

1000

1250

1500

1750

2000

2250

Days Post Tumor Implantation

Tu

mo

r V

olu

me, m

m3

PBS

Free C/EBP ASO

exoASO C/EBP

anti-PD1

exoASO Scramble

exoASO C/EBP + PD1

Free C/EBP ASO + PD1

anti-CSF1R

0

250

500

750

1000

1250

1500

1750

2000

2250

exoASO Scramble PBS

Free C/EBP ASOexoASO C/EBP

anti-PD1

Free C/EBP ASO

+PD1exoASO C/EBP

+PD1

anti-CSF1R

(a) Exo Only

(b) Exo+ASO

(c)Exo+

Chol-ASO

D0

CT26, SC

mABsIP injection, DIW

D8 D1960-75mm3

D27→

ExoASO, free ASOIT injection, TIW

Cholesterol-tagged ASO (Chol-ASO) shows efficient loading on exosomes, and high uptake in

macrophages. A: Exosome pellets in ultracentrifuge tubes, post loading and clean up: (a) exosomes only,

(b) exosomes + ASO-Cy5, (c) exosomes + Chol-ASO-Cy5. B: Quantification of Chol-ASO by Ribogreen

Assay on either WT or PTGFRN over-expressing exosomes. C: Uptake of equivalent doses of either

exoASO-Cy5 or freeASO-Cy5 in M0 and M2 polarized primary human macrophages.

Quantification of ASOs on exosomes

Effective knockdown of C/EBPβ and reduction of CD163 expression in vitro by exoASO. A: Human

primary M2 macrophages were incubated for 48 hours with equivalent doses of exoASO and free ASO

targeting C/EBPβ, along with an exoASO scramble control. Gene expression levels of C/EBPβ (A) and the

M2 marker CD163 (B) were analyzed by qPCR, post treatment. One representative donor of five is shown

A B

exoASO C/EBPβ IC50 427.1 nM

Free C/EBPβ ASO IC50 982.0 nM

exoASO C/EBPβ IC50 263.9 nM

Free C/EBPβ ASO IC50 525.7 nM

Effective M2 to M1 macrophage reprogramming in vitro by exoASO. Human primary M2 macrophages

were incubated for 48 hours with equivalent doses of exoASO and free ASO targeting C/EBPβ, along with

an exoASO scramble control. A: Gene expression analysis was performed by Nanostring using the

nCounter Human Myeloid Innate Immunity Panel v2. One representative donor out of three is shown. B:

Cytokine production was analyzed after 24 hours treatment with LPS (10ng/ml) using a multiplex flow

cytometry assay (LegendPlex). One representative donor out of four is shown. ****, P < 0.0001, ***, P <

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

Exosome tropism to myeloid cells promotes selective delivery of ASOs. A-C: 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, liver (A), spleen (B) and peripheral blood (C) were collected and

analyzed by flow cytometry. D: CT26 SC tumors received one intratumoral (IT) dose (4μg) of fluorescently-

labeled Cy5 exoASO. One hour later, tumors were dissected and enzymatically digested, and tumor cell

suspensions analyzed by flow cytometry.

A

B

C

D

In vivo knockdown by exoASO. CT26 tumors were treated IT with 4μg of exoASO C/EBPβ or exoASOScramble, 3 injections (q.o.d.). After treatment, tumor-associated myeloid cells were isolated using CD11b-positive selection magnetic bead isolation. A-B: C/EBPβ (A) and Arg-1 (B) knockdown in pre- and post-enrichment samples were analyzed by qPCR in whole tumor and in tumor CD11b+ cells. *, P < 0.05, **, P <0.01 and ***, P < 0.001 by unpaired two-tailed t-test comparing exoASO C/EBPβ and exoASO Scramble

In vivo reprogramming of TAMs by exoASO. CT26 tumors were treated IT with 4μg of exoASO C/EBPβor exoASO Scramble, 3 injections (q.o.d.). After treatment, tumor-associated myeloid cells were isolatedusing CD11b-positive selection magnetic bead isolation. Gene expression analysis in the enriched myeloidfraction was performed by Nanostring using the nCounter Human Myeloid Innate Immunity Panel v2.

Anti-tumor activity of exoASO C/EBPβ. CT26 tumor cells were implanted SC in the flanks of mice (n = 10per group). ExoASO and free ASO were dosed IT and antibodies intraperitoneally following dosing regimenin (A). A: Dosing scheme. B-C: Tumor volumes of testing agents. Geometric means of tumor volumes aredepicted in (B). Individual tumor growth curves are shown in (C). CR: Number of complete responses.

A B

B C

A

•exoASO is a novel, engineered exosome that can selectively deliver antisense oligonucleotides to tumor

associated M2 macrophages.

•exoASO enables selective silencing of C/EBPβ, a key transcription factor that controls the

immunosuppressive program.

•Effective silencing of C/EBPβ both in vitro and in vivo, lead to the modulation of key factors involved in the

M2→M1 transition

• Intratumoral administration of exoASO C/EBPβ resulted in potent monotherapy anti-tumor responses,

resulting in 60% complete responses via IT route of administration.

•Reprogramming of tumor associated myeloid cells by targeting C/EBPβ resulted in vastly improved anti-

tumor outcomes, as compared to anti-CSF1R, a macrophage-depleting therapy

•Collectively, exoASOs against C/EBPβ represent a first-in-class strategy to target tumor-associated

myeloid cells in a highly selective manner.

Induction of pro-inflammatory M1 markers following exoASO treatment, by CIVO®. YUMM1.7 tumorswere dosed with IT microinjections of exoASO C/EBPβ, Free C/EBPβ ASO, or exoASO Scramble. Micewere euthanized 24 hours after one single dose, (n=6 mice per group). A: Schematic for the Comparative InVivo Oncology (CIVO®) Platform by Presage Biosciences. B: Expression of TNFα, CD11b, iNOS and F4/80by In situ hybridization. Each panel row is a different injection site on the same tumor. FTM: fluorescenttracking marker denoting the injection site.

*

********

********

*******

****** ********

****

CIVO Microinjection

Image Analysis

CIVO

tumor

TNFα DAPI FTM CD11b DAPI FTM iNOS DAPI FTM F4/80 DAPI FTM

ex

oA

SO

C/E

BPβ

Fre

e

C/E

BPβ

ASO

ex

oA

SO

Sc

ram

ble

A B

6/10 CR 8/10 CR

0/10 CR

A

B

A B C

GENE EXPRESSION ANALYSIS

8.2x 3.5x 0.15x0.03x

9x

3.4x2.2x

2.7x

12x

11x

11x

0.2x0.08x

0.16x3.8x