Poster #A18
Tumor Immunology and Immunotherapy
Imprime PGG, a soluble yeast b-glucan PAMP, synergizes with anti-PD-1 antibody to enhance CD8 T cell anti-tumor immunity
Immune checkpoint inhibitor (CPI) therapy has provided compelling, durableanti-tumor T cell immunity for some cancer patients, particularly those with pre-existing anti-tumor T cell responses. Unfortunately, the majority of patients showlittle to no pre-existing anti-cancer T cell response and do not benefit from singleagent CPI therapy. For these patients, novel therapeutic approaches thatinitiate or revitalize the anti-cancer T cell responses may be particularlypromising as combination agents with CPIs as these would provide theactivated T cell population upon which CPIs would act.
Proper activation of the innate immune system is essential to generaterobust immune responses. Central to this process are non-self danger signalsknown as Pathogen-Associated Molecular Patterns (PAMPs) that bind to andsignal via conserved pattern recognition receptors (PRRs) expressed by innateimmune effector cells (macrophages, monocytes, dendritic cells, neutrophils).Imprime PGG is a soluble yeast b-1,3/1,6 glucan PAMP currently in multipleclinical trials in combination with pembrolizumab (anti-PD-1). Unlike otherPAMPs that must be locally administered to avoid systemic cytokine storms,Imprime has been administered safely by intravenous infusion to >400 humansubjects (>325 cancer patients). Mechanistically, here we show that Imprimeactivates innate immune cells by binding the PRR dectin-1, eliciting dendriticcell and monocyte expression of co-stimulatory ligands, mobilization of myeloidcells, as well as production of a variety of chemokines and cytokines, includingtype I interferon. To examine if Imprime’s innate modulating effects alter CD8 Tcell priming, we used the ovalbumin (OVA)—specific OT-I CD8 T cell system.Following OT-I adoptive transfer into congenic C57BL/6 recipients, mice wereimmunized with OVA peptide and Imprime. Co-administration of Imprimeincreased OT-I expansion compared to peptide alone and induced theirdifferentiation into polyfunctional Tbet+ effector cells. Based on Imprime’s abilityto link innate and adaptive immune responses, we asked if Imprime wouldenhance the anti-tumor efficacy of anti-PD-1 antibody therapy. C57BL/6 micewere injected s.c. with the murine adenocarcinoma cell line MC38. Once tumorsreached ~50mm3, mice were randomized to treatment groups: vehicle, Imprime(1.2mg twice weekly), anti-PD-1 antibody (RMP1-14 clone, 100µg twiceweekly), or Imprime + anti-PD-1 antibody. While anti-PD-1 treatment alonesignificantly reduced tumor growth compared to vehicle or Imprime groups,combination treatment provided superior tumor control with a statistically higherfrequency of mice completely clearing tumor in the combination groupcompared to the anti-PD-1 group (4/57 vs 14/60, respectively). To assess theimmunobiology elicited by treatment, tumors were harvested after two weeks oftherapy, and tumor-infiltrating leukocytes were stimulated with PMA/ionomycinor tumor-specific peptides. Relative to other treatment groups, CD8 TIL fromcombination-treated mice had superior frequencies of cells capable ofproducing IFN-g, TNF-a, or IL-2. Furthermore, mice from these MC38 studiesthat had previously cleared their tumors were protected against tumor re-challenge in the opposite flank, demonstrating that these mice had durable andprotective adaptive immunity. Together, our data demonstrate that Imprime PGGis a unique, clinically relevant PAMP that can be administered systemically toactivate key innate immune cell populations and to drive T cell based anti-cancer responses. Consequently, Imprime PGG synergizes with anti-PD-1antibody treatment to provide anti-tumor CD8 T cells with superior effectorfunctions. We are currently working to extend these mechanistic findings in ourongoing phase II clinical trials.
Background
Spleen 24hrs
Neutrophils
Ly6Chi monocytes cDC1 (XCR1+)
cDC2 (CD172a+)
Vehicle
Imprime
Imprime
b cDC1 (XCR1+)Migratory
ResidentNeutrophils
Ly6Chi monocytes
Imprime
LCs
skin-draining LNs 24hrscDC2 (CD172a+)
CD11bnegCCR7+ CD11b+CCR7+
CD11b+CCR7neg
Vehicle
Imprime
c
Figure 1. Imprime binds to myeloid lineage cells
Figure 1. Imprime binds to myeloid lineage cells. Naïve C57BL/6 mice were injected with 1.2mg Imprime i.v. and various myeloid populations were examine 24hrs later for Imprime binding. (A) Identification of DC subsets. Ly6Chi monocytes and neutrophils were first gated out and then the bulk DC population was then identified as CD11c+MHC-II+Lineageneg. DC subsets were then further subset based on expression of CD172a (Sirp-a), XCR1, CD24, and CCR7. (B) Imprime binding profiles 24hrs post injection in the spleen (B) and skin-draining LNs (C). Plots are representative of 3-4 experiments with at least n = 3 per experiment.
FSC
SSC
CD11b
Ly6C
MHC II
Line
age
CD11c
MH
C II
CD172a
XCR
1
CCR7
CD
24
CD24
CC
R7
CD11b
CC
R7
Boolean gating:NOT monocytes and
neutrophilsidentification of
total DCs
cDC1
resident cDC1
dermal (CD103+)migratory
cDC1
cDC2
Langerhans cells
total cDC2
CD11b+
resident
CD11b+
migratory
CD11bneg
migratory
Ly6G
Ly6C
Ly6Chi mono
neutrophils
CD11b
Dum
p
aA
Spleen 24hrs
Neutrophils
Ly6Chi monocytes cDC1 (XCR1+)
cDC2 (CD172a+)
Vehicle
Imprime
Imprime
b cDC1 (XCR1+)Migratory
ResidentNeutrophils
Ly6Chi monocytes
Imprime
LCs
skin-draining LNs 24hrscDC2 (CD172a+)
CD11bnegCCR7+ CD11b+CCR7+
CD11b+CCR7neg
Vehicle
Imprime
c
Figure 1. Imprime binds to myeloid lineage cells
Figure 1. Imprime binds to myeloid lineage cells. Naïve C57BL/6 mice were injected with 1.2mg Imprime i.v. and various myeloid populations were examine 24hrs later for Imprime binding. (A) Identification of DC subsets. Ly6Chi monocytes and neutrophils were first gated out and then the bulk DC population was then identified as CD11c+MHC-II+Lineageneg. DC subsets were then further subset based on expression of CD172a (Sirp-a), XCR1, CD24, and CCR7. (B) Imprime binding profiles 24hrs post injection in the spleen (B) and skin-draining LNs (C). Plots are representative of 3-4 experiments with at least n = 3 per experiment.
FSC
SSC
CD11b
Ly6C
MHC II
Line
age
CD11c
MH
C II
CD172a
XCR
1
CCR7
CD
24
CD24
CC
R7
CD11b
CC
R7
Boolean gating:NOT monocytes and
neutrophilsidentification of
total DCs
cDC1
resident cDC1
dermal (CD103+)migratory
cDC1
cDC2
Langerhans cells
total cDC2
CD11b+
resident
CD11b+
migratory
CD11bneg
migratory
Ly6G
Ly6C
Ly6Chi mono
neutrophils
CD11b
Dum
p
a
B
cDC1 r
eside
nt
cDC1 m
igrato
ry
cDC2 C
D11b- m
igrato
ry
cDC2 C
D11b+ m
igrato
ry
cDC2 C
D11b+ re
siden
tLC
s0.0
0.5
1.0
1.5
2.0
2.5
Fold
cha
nge
in M
FI
over
veh
icle
CD86
* *
***
*****
MFI
fold
cha
nge
over
veh
icle
cDC1 r
eside
nt
cDC1 m
igrato
ry
cDC2 C
D11b- m
igrato
ry
cDC2 C
D11b+ m
igrato
ry
cDC2 C
D11b+ re
siden
tLC
s0.0
0.5
1.0
1.5
2.0CD40
***
**
Imprime matures DCsC
cDC1 r
eside
nt
cDC1 m
igrato
ry
cDC2 C
D11b- m
igrato
ry
cDC2 C
D11b+ m
igrato
ry
cDC2 C
D11b+ re
siden
tLC
s0
1
2
3PD-L1
***
**
***
*****
CD86 CD40 PD-L1
D
Ly6C
hi mon
o
Neutro
phils
Ly6C
hi mon
o
Neutro
phils
Ly6C
hi mon
o
Neutro
phils
0
5
10
15
20
1
Fold
cha
nge
over
veh
icle Spleen
LNBlood
** ***
***
***
* ***
Ly6C
hi mon
o
Neutro
phils
0
5
10
15
20
1
Fold
cha
nge
over
veh
icle
Dectin-1-/-WT
***
***
Imprime mobilizes and matures inflammatory Ly6Chi monocytes
Figure 1. Imprime binds to and activates myeloid lineage cells. Naïve C57BL/6 mice were injected with 1.2mg Imprime i.v. and various myeloid populations in skin-draining LNs (sdLNs) were examine 24hrs later for Imprime binding. (A) Identification of DC subsets in skin-draining LNs. Ly6Chi monocytes and neutrophils were first gated out and then the bulk DC population was then identified as CD11c+MHC-II+Lineageneg. DC subsets were then further subset based on expression of CD172a (Sirp-a), XCR1, CD24, and CCR7. (B) Imprime binding profiles 24hrs post injection in the sdLNs and (C) expression levels of CD86, CD40, and PD-L1. (D) Imprime causes Dectin-1-dependent mobilization of Ly6Chi monocytes in sdLNs (E) and upregulation of activation and co-stimulatory molecules.
CD11c MHC II
CD86 CD40 PD-L1
PBSImprime
ELy6Chi monocytes
LNs 24hrs post Imprime
Peripheral LNs 16hrs post Imprime i.v.
Cd274Cd40Cd80
Clec7a
0
1
2
3
4
5
Rel
ativ
e m
RN
A ex
pres
sion
*
**
*
Ifng
Tnfa Il1a
Il1b Il6
Il23a
(p19
)
Il12b
(p40
)0
4
8
12
16
202040
Rel
ativ
e m
RN
A ex
pres
sion
**
*
*
*
**
Ccl2Ccl3Ccl4Cxcl1Cxcl2
0
5
10
15
20
Rel
ativ
e m
RN
A ex
pres
sion
*
*
*
*
*
Irf5Irf7Irf9
Socs1
Socs3Stat1Stat2Stat3
0
1
2
3
4
5
Rel
ativ
e m
RN
A ex
pres
sion
*
*
*
*
*
**
*
Mx1
Oas1a
Oasl1
Usp18
0
2
3
4
1
Rel
ativ
e m
RN
A ex
pres
sion
**
**
* *
WTDectin-1-/-
Ifnar1-/-
Activation-associated Cytokines Chemokines
Signaling IFN stimulated genesImprime binds to antigen-presenting cells
Imprime induces a Dectin-1-dependent pro-inflammatory profileA
B
PBS
Impri
me0
100
200
300
400
500
pg/1
0mg
tissu
e
TNF-α
***
PBS
Impri
me0
200
400
600
800
1000
pg/1
0mg
tissu
e
IFN-γ
*
PBS
Impri
me0
20
40
60
80
pg/1
0mg
tissu
e
GM-CSF
***
PBS
Impri
me0
500
1000
1500
2000
pg/1
0mg
tissu
e
IL-6
**
PBS
Impri
me0
20
40
60
80
100
pg/1
0mg
tissu
e
IL-1β
**
PBS
Impri
me0
1000
2000
3000
4000
5000
pg/1
0mg
tissu
e
CCL2*
PBS
Impri
me0
100
200
300
400
pg/1
0mg
tissu
e
CCL3***
PBS
Impri
me0
50
100
150
pg/1
0mg
tissu
e
CCL4
*
PBS
Impri
me0
10
20
30
40
pg/1
0mg
tissu
e
CXCL2
*
In vivo cytokine production in LNs: 24hrs post Imprime
Figure 2. Imprime induces Dectin-1-dependent production of pro-inflammatory cytokines and chemokines. (A) Naïve WT or Dectin-1-/- C57BL/6 mice were injected with PBS or 1.2mg Imprime i.v. and 16hrs later skin-draining lymph nodes were harvested and analyzed for transcriptional changes using a custom QuantiGene Plex. Identified genes were categorized into Activation-associated, Cytokines, Chemokines, Signaling and IFN-stimulated genes. IFNaR1-/- mice were used to confirm the dependence of ISG’s on type I IFN. (B) Naïve C57BL/6 mice were injected with PBS or 1.2mg Imprime i.v. Skin-draining LNs were harvested 24hrs later and cell lysates were analyzed for cytokine and chemokine proteins using the Luminex platform.
OVA peptide +/- Imprime (1.2mg)
Exp 9996
Transfer 1x105 naïve OT-I CD8 T cells specific to OVA257-264 peptide
C57BL/6 Day 7 Harvest spleen/LN Assess OT-I response
(peptide and Imprime are NOT conjugated)
WT Dectin-1-/-0
1
2
3
4
5
% O
T-I o
f tot
al C
D8
d5 PBL
PBSImprimeLPS
*** ***
ns
**
Statistics were done using a one-way ANOVA, Kruskal-Wallis nonparametric test (because of combining multiple experiments there were significant differences in stdev using a parametric test).
WT Dectin-1-/-0.0
0.5
1.0
1.5
% O
T-I o
f tot
al C
D8
d7 spleen PBSImprimeLPS
******
ns
**
Statistics were done using a one-way ANOVA, Kruskal-Wallis nonparametric test (because of combining multiple experiments there were significant differences in stdev using a parametric test).
Day 5 blood Day 7 spleen
Figure 3. Imprime enhances expansion and effectordifferentiation of antigen-specific CD8 T cells. (A)Vaccination model. Naïve WT or Dectin-1-/- mice received OT-ICD8 T cells. The following day they were injected with 100µgOVA257-264 peptide i.v. and either PBS, Imprime, or LPS. Themagnitude of the OT-I response was assessed on day 5 in theblood (B) and day 7 in the spleen (C). (D) OT-I CD44 and Tbetexpression on day 7 in the spleen. (E) On day 7, splenocyteswere stimulated with OVA peptide for 5hrs with brefeldin A. Cellswere then stained for intracellular IFN-g, TNF-a, and IL-2.
A
B C
E
PBS
Impri
meLP
S0
20
40
60
80
100
% o
f OT-
I
SPDP
none
TP
*** **
***
*****
***ns
PBS
Impri
meLP
S0
20
40
60
80
100
% o
f OT-
I
SPDP
none
TP
*** **
***
*****
***ns
Cytokine production:combinations of IFN-g, TNF-a, IL-2
none1 cytokine2 cytokines3 cytokines
% o
f OT-
I
D
Tbet
CD
44
+PBS +Imprime
HostOT-I
Vehicl
e
Impr
ime
PD1
Impr
ime+
PD10
10
20
30
40
50
% m
ice
with
no
palp
able
tum
or
4/57
14/60
1/57 0/59
**
Figure 4. Imprime synergizes with anti-PD-1 antibody to reduce MC38 tumor growth. C57BL/6 mice were injected with MC38 s.c. When tumors were ~50mm3, mice were treated with PBS (Vehicle), Imprime, anti-PD-1 (clone RMP1-14), or anti-PD-1 + Imprime. (A) Tumor growth kinetics. Mean tumor volume is shown without error bars. (B) Tumor volumes on d21 post tumor inoculation. Each symbol represents a single mouse and data is shown as mean +/- SEM. (C) Percentage of MC38 tumor-bearing mice that cleared tumor by d21 post inoculation. Fractions show the number of mice without palpable tumors out of the total number of mice in each treatment group pooled from 3 experiments. (D) Comparison of granzyme B expression by CD8 TIL after 2 weeks of treatment. (E) TIL were isolated from MC38 tumors after 14 days of treatment and stimulated with PMA/ionomycin. Tumor size versus % CD8 IFN-g, TNF-a, or IL-2 production was plotted for each individual mouse. Linear correlations (solid lines) and Pearson correlations were calculated for each treatment group.
Imprime+PD1
Imprime
PD1
Vehicle
DAPICD8GrzB
Vehicle
ImprimePD1
Combo
0
20
40
60
% o
f CD
3
CD8+GrzB+
**
Fig. 6d
Imprime+PD1
Imprime
PD1
Vehicle
DAPICD8GrzB
Vehicle
ImprimePD1
Combo
0
20
40
60
% o
f CD
3
CD8+GrzB+
**
Fig. 6dVehicle Imprime
PD-1 Imprime + PD-16 9 12 15 18 21
0100200300400500600700
Day post tumor
Mea
n Tu
mor
Vol
ume
(mm
3 )
VehicleImprimePD1Imprime+PD1
day post tumortreatment initiation Veh
icle
Impr
ime
PD1
Impr
ime+
PD10
200
400
600
800
1000
1200
Tum
or V
olum
e (m
m3 )
*
A B
C
D
0 500 1000 15000
20
40
60
80
100
tumor mm3
% IF
N-γ
+ of
CD
8 TI
L(P
MA
/iono
myc
in)
VehicleImprimePD-1Combo
0 500 1000 15000
10
20
30
40
50
tumor mm3
% IL
-2+
of C
D8
TIL
(PM
A/io
nom
ycin
)
0 500 1000 15000
20
40
60
80
100
tumor mm3
% T
NF-α
+ of
CD
8 TI
L(P
MA
/iono
myc
in)
IFN-g TNF-a IL-2E
A general structure of yeast-derived Imprime
PGG
Conclusions• Imprime binds to a wide variety of professional
antigen-presenting cells (APCs).• Imprime matures APCs via Dectin-1, initiating
production of localized pro-inflammatory cytokines • Imprime enhances CD8 T cell expansion and their
capacity to produce effector cytokines.• Imprime synergizes with anti-PD-1 antibody
therapy in the murine MC38 tumor model. Combination therapy results in increased clearance of established tumors, increased granzyme B+ CD8 TIL, and improved TNF-a/IL-2 production by CD8 TIL.
• These data provide rationale for Imprime and checkpoint inhibitor therapy which is currently being tested in clinical trials.
Ross B. Fulton, Steven M. Leonardo, Kathryn A. Fraser, Takashi O. Kangas, Adria B. Jonas, Anissa S.H. Chan, Nandita Bose, Keith B. Gorden, Jeremy R. Graff, and Mark Uhlik. Biothera Pharmaceuticals, Inc., Eagan MN, 55121 [email protected]