powerpoint presentation...exo-aso m0 free aso-m0 exosome mediated delivery of antisense...
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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