Con trala tera l tum or s...Contralateral tumor Injected tumor-50 0 50 100 150 200 0.0 0.2 0.4 0.6...
Transcript of Con trala tera l tum or s...Contralateral tumor Injected tumor-50 0 50 100 150 200 0.0 0.2 0.4 0.6...
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