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10 18 26 34 42
90
100
110
120
Days after cell injection
%
in
Bo
dy
We
igh
t +
SE
M
10 18 26 34 42
0
1
2
3
Days after cell injection
Me
an
Sto
ol S
co
re +
SE
M
10 18 26 34 42
0
1
2
3
4
5
6
Days after cell injection
Mean
DA
I +
SE
M
Vehicle (no colitis), Day 0-41
Fc control, Day 0-41
ALPN-101, Day 0-41
ALPN-101, Day 0 (single dose)
ALPN-101, Day 14 (single dose)
ALPN-101, Day 14-41
Acknowledgements
Figure 5: ALPN-101 Treatment in the CD4+CD45RBhigh T Cell-Induced Mouse
Model of Colitis
• T cell costimulation is strongly implicated in the pathogenesis of IBD, yet CD28 costimulatory pathway inhibitors (e.g. abatacept) have not
proven clinically efficacious, implicating an alternative costimulatory pathway.
• CD28 predominates in naïve T cells and is less critical in activated, effector T cells. In contrast, costimulatory receptor ICOS (Inducible T cell
Co-Stimulator) is upregulated and mediates costimulation in post-activation T cells - suggesting ICOS may be more relevant in active disease.
• ALPN-101 (ICOSL vIgD-Fc) is an Fc fusion protein of a human inducible T cell costimulator ligand (ICOSL) variant immunoglobulin domain
(vIgDTM) engineered to inhibit both CD28 and ICOS.
• ALPN-101 has potent in vitro immunosuppressive activity and in vivo efficacy in models of disease for which both CD28 and ICOS have been
implicated (aGvHD, RA, Sjögren’s, Lupus, MS).
• Here, we demonstrate potent activity of ALPN-101: (1) in vitro using PBMC from Crohn’s and ulcerative colitis patients, demonstrating superior
suppression of T cell activation and cytokine release, and (2) in vivo in a mouse T cell transfer model of chronic colitis, showing its efficacy to
both prevent and treat disease.
ALPN-101, A FIRST-IN-CLASS DUAL ICOS/CD28 ANTAGONIST, DEMONSTRATES EFFICACY IN
PATIENT-DERIVED PBMC IN VITRO AND IN AN IN VIVO T CELL TRANSFER MODEL OF CHRONIC
INFLAMMATORY BOWEL DISEASE (IBD)
Introduction
• ALPN-101 (ICOSL vIgD-Fc), a novel therapeutic candidate for inflammatory disease, is a dual CD28 and ICOS
T cell co-stimulation pathway inhibitor that targets both naïve and activated pathogenic T cells, including ICOS+
cells that may escape inhibitors that target only the CD28 pathway
• ALPN-101 inhibits cytokine production in vitro from human colitis patient PBMC more potently than single CD28
or ICOS pathway inhibitors
• ALPN-101 demonstrates effector memory T cell and cytokine suppression in mouse in vivo translational models
of inflammatory bowel disease, and appears to completely prevent development of colitis even with delayed
repeat dose administration. Single dose administration at day 0 or day 14 still resulted in
milder colitis compared to Fc control.
• A Ph1 healthy volunteer study to evaluate safety and pharmacodynamic activity of single
and multiple intravenous and subcutaneous escalating doses of ALPN-101 has recently
been completed (NCT03748836). Therapeutic studies in inflammatory diseases, including
acute graft-versus-host disease (NCT04227938, BALANCE; Yang 2019), are in preparation.
P156 CROHN’S & COLITIS CONGRESS » JANUARY 23-25, 2020 » AUSTIN, TX, USA
Figure 2: ALPN-101 Binds CD28 and ICOS and Prevents Ligand Binding
Figure 4: Superior Inhibition of Cytokine Secretion from Stimulated Patient
PBMCs with ALPN-101Figure 6: ALPN-101 Significantly Reduces Disease in the CD4+CD45RBhigh
T Cell-Induced Mouse Model of Colitis
Summary and Conclusions
AlpineImmuneSciences.com @AlpineImmuneSci © 2020 Alpine Immune Sciences. All rights reserved.
Stacey R. Dillon, Lawrence S. Evans, Susan Bort, Sherri Mudri, Katherine E. Lewis, Mark Rixon, Janhavi Bhandari, Kayla Kleist, Stanford L. Peng, and Kristine M. SwiderekAlpine Immune Sciences, Inc. Seattle, WA
Figure 1: ALPN-101 Blocks Both CD28 & ICOS T Cell Costimulation Pathways
ALPN-101 inhibits both
CD28- and ICOS-mediated
costimulation. In contrast,
abatacept (BMS) inhibits
only the CD28/CTLA-4 -
CD80/CD86 axis, while
prezalumab (anti ICOSL
mAb, Amgen) inhibits only
the ICOS pathway.
CD28 costimulation is critical
for naïve and memory CD4+
T cell activation. ICOS
signaling augments CD28
costimulation but also plays
non-redundant roles; ICOS
deficiency leads to defective
humoral responses in both
mice and humans.
CD28 inhibition ICOS-only inhibitionCD28+ICOS dual blockade
CD80/86(B7)
CD28
T cell
prezalumab
ICOSL
ICOS
T cellactivation
APC/B cell
CD80/86(B7)
CD28
CTLA4-lg
T cellactivation
ICOSL
ICOS
ICOSL
ICOS
CD80/86(B7)
CD28
T cell Inactivation
ALPN-101
A. ALPN-101 titrated and incubated with CHO cells expressing human CD28 or ICOS; bound protein detected with anti-human IgG-PE and measured
by flow cytometry.
B. ALPN-101 or comparators titrated and incubated with fixed amounts of labeled CD86 or ICOSL and added to CHO cells expressing human CD28 or
ICOS; binding measured by flow cytometry.
C. CD28 blockade demonstrated by inhibition of artificial APC expressing OKT3 and human CD86 stimulating CD28+ Jurkat/IL-2 cells (IL-2 promotor
driven luciferase expression; Promega). ICOS blockade demonstrated by inhibition of aAPC expressing OKT3 and human ICOSL stimulating ICOS+
Jurkat/IL-2 cells (transduced with a chimeric molecule consisting of the extracellular domain of human ICOS and the intracellular domain of CD28).
D) Ligand Binding IC50 (nM)A) Target Binding B) Blockade of Ligand
Binding to Target ReceptorC) Costimulation Blockade
10 100 1000 10000 1000001000000
0
10000
20000
30000
40000
50000
CD28
[ pM ]
Med
ian
Flu
ore
scen
ce
WT ICOSL
ALPN-101
Abatacept
Belatacept
Prezalumab
100 1000 10000 100000 1000000500
1000
1500
2000
CD86 CD28
[ pM ]
Med
ian
Flu
ore
scen
ce
ALPN-101
Abatacept
Belatacept
10 100 1000 10000 1000001000000
0
20000
40000
60000
ICOS
[ pM ]
Med
ian
Flu
ore
scen
ce
WT ICOSL
ALPN-101
Abatacept
Belatacept
Prezalumab
100 1000 10000 100000 10000000
25000
50000
75000
ICOSL ICOS
[ pM ]
Med
ian
Flu
ore
scen
ce
WT ICOSL
Prezalumab
ALPN-101
1 100 10000 1000000700
1050
1400
1750
2100
ICOS Costimulation Blockade
[ pM ]
RL
U
Prezalumab
ALPN-101
1 100 10000 10000000
3000
6000
9000
CD28 Costimulation Blockade
[ pM ]
RL
U
Belatacept
ALPN-101
AbataceptCell Surface
Expressed TargetCD28 ICOS
Ligand CD80 CD86 ICOSL
Inh
ibit
or
WT ICOSL - - 17.2
Prezalumab - - 23.3
Abatacept 5.4 12.9 -
Belatacept 2.5 2.8 -
ALPN-101 1.0 0.7 4.3
A) In Vitro PBMC stimulation assay
• Crohn’s Disease (CD) or Ulcerative Colitis (UC) patient
PBMC were stimulated at a 20:1 ratio with artificial APC
[fixed K562 expressing cell surface OKT3 (anti-CD3),
CD80, CD86, ICOSL]
• Antagonists were added at 100 nM
• After 48 h, supernatants were collected and assayed for
cytokine concentrations
Cohort n Name Cells injected Dosing Regimen (IP, 0.1 ml))
1 12 Vehicle (no colitis) Day 0-41 CD4+ Vehicle - 2x/week: Day 0 – Day 41
2 16 Fc1.1 Day 0-41
CD4+CD45RBhigh
Fc control 260 ug 2x/week: Day 0 – Day 41
3 12 ALPN-101 Day 0-41 ALPN-101 400 ug 2x/week: Day 0 – Day 41
4 12 ALPN-101 Day 0 ALPN-101 400 ug 1x on Day 0*
5 12 ALPN-101 Day 14 ALPN-101 400 ug 1x on Day 14*
6 12 ALPN-101 Day 14-41 ALPN-101 400 ug 2x/week: Day 14 – Day 41*
Treatment Groups
The authors thank the team at Hooke Laboratories, Inc. (Lawrence, MA) for conducting the colitis study, and our Alpine colleagues for their contributions to this work.
C.B17 (SCID)
male recipients
Treat with ALPN-101 or Fc control
Collect blood for flow analysis
on Days 13, 20, and 27
BALB/c male
spleendonors
• Colon weight (mg)/length (mm)
• Colon & small intestine (histology)
• Distal 1/3 of colon cultured to assess
cytokine secretion ex vivo
• Blood, mesenteric LN for flow analysis
• Serum (PK, cytokines)
Day 0 Days 1-41
Sort CD4+ &
CD4+CD45RBhigh
(Treg-depleted)
cells
ALPN-101 (0.4 mg) IP
0.3 million
cells IV
(A) % Change in Body Weight (B) Stool Scores (C) Disease Activity Index
ScoreClinical observations
Stool Body Weight / Day 0 weight
0 Stool fairly compact with no signs of mucus. When pressed, keeps shape, does not fall apart ≥ 99%
1 Stool soft, no signs of mucus. When pressed, compacts easily, then breaks apart; OR No stool, but a clear liquid can be seen [95-99%)
2 Very soft stool, not quite diarrhea; OR Wet pellet is formed, falls apart when pressure applied; OR No stool, but colored mucus visible [90-95%)
3 Mucus and some diarrhea present. Stool does not hold shape when touched; OR No stool, but some signs of diarrhea [85-90%)
4 Diarrhea is present; OR No stool, but excessive signs of diarrhea < 85%
Scoring System: Disease Activity Index (DAI): Body Weight Score + Stool Score
B) Cytokine Secretion from Stimulated PBMC C) Percent Inhibition of Cytokine Secretion Relative to Fc Control
IC50 values calculated using GraphPad Prism
Days 42-43: Study Termination
T cell
CD28
ICOS
Artificial APCK562/OKT3/CD80/CD86/ICOSL
CD80/86
ICOSL
OKT ScFv
Target(s)
ICOSLPrezalumab
(anti-ICOSL mAb)
CD80/
CD86Abatacept
(CTLA-4-Fc)
ALPN-101CD28
ICOS
ICOSWT ICOSL IgV
Antagonists
B. Cytokines secreted from stimulated PBMC from Crohn’s Disease (CD) or Ulcerative Colitis (UC) patients were analyzed by
ELISA or Milliplex® (EMD Millipore)
C. % Inhibition determined using the following formula: [(Fc control value – Exp value)/Fc control value)]*100. For most
analytes, ALPN-101 demonstrated greater cytokine inhibition than observed with abatacept or prezalumab alone or combined
(i.e. IL-17A)
Pathway
(Treatment)Cytokine
IL-2
IFNG
IL-3
IL-6
IL-12p70
IL-4
IL-5
GM-CSF
IL-13
IL-17RA
IL-17A
TNF-α
IL-1β
IL-1α
IL-12p40
VEGF
IL-2
IFNG
IL-3
IL-6
IL-12p70
IL-4
IL-5
GM-CSF
IL-13
IL-17RA
IL-17A
TNF-α
IL-1β
IL-1α
IL-12p40
VEGF
IL-2
IFNG
IL-3
IL-6
IL-12p70
IL-4
IL-5
GM-CSF
IL-13
IL-17RA
IL-17A
TNF-α
IL-1β
IL-1α
IL-12p40
VEGF
Crohn's Disease Ulcerative Colitis
ICOS Only
(Prezalumab)
CD28 Only
(Abatacept)
CD28 / ICOS
(ALPN-101)
% Inhibition
vs. Fc
Histology Score Criteria
Mucosa1
0 Normal
1 3–10 IEL/HPF2. Focal damage3
2 >10 IEL/HPF. Rare crypt abscesses
3 >10 IEL/HPF. Multiple crypt abscesses, erosion/ulceration
Submucosa
0 Normal, widely scattered leukocytes
1 Focal aggregates of leukocytes
2 Leukocyte infiltration with expansion of submucosa
3 Diffuse leukocyte infiltration
Muscularis
0 Normal, widely scattered leukocytes
1 Widely scattered leukocyte aggregates between muscle layers
2 Leukocyte infiltration with focal effacement of the muscularis
3 Extensive leukocyte infiltration with transmural effacement of muscularis1 Epithelium and lamina propria2 IEL, intraepithelial lymphocytes; HPF, high power field3 Immature enterocytes, increased mitotic figures
Colon Histology Scoring
Efficacy of ALPN-101 in a murine T cell transfer model of colitis (Fig. 5), using various dosing regimens, was evaluated based on
the improvement of the disease activity index (A-C), suppression of T cells in blood and mesenteric lymph nodes (D), suppression
of pro-inflammatory cytokines in serum (E), and macroscopic and microscopic assessment of the colon post mortem (F).
(F) Macroscopic and Microscopic Assessment of the Colon
Figure 3: ICOS/CD28 Role in Inflammatory Bowel Disease
CD28+ ICOS+ ICOS+
ICOS+
Key T cell subsets - Activated
- Memory- Effector- Follicular Helper T cells
APC
T cell activationICOSCD28
(some cells)
NaïveT cell
CD28+
ICOS+ T cells escape inhibition with
CD28 –CD80/CD86 pathway blockers
(e. g. abatacept)
IndicationPathogenic
Cell Type
Clinical Effect of
CD28 Pathway
Inhibition
Potential Role for ICOS in Disease Pathogenesis
Crohn’s Disease
(CD)T cells
Not efficacious
in moderate to
severe CD 1
ICOS expression & CD4+ ICOS+ lamina propria mononuclear cells
(LPMC) elevated in inflamed mucosa compared to non-inflamed or
PBMCs 2
ICOS expression on CD4+ LPMC directly correlates with endoscopic
disease activity scores (CDAI > 150)
Blocking ICOS on CD28-deficient T cells in a CD45RBhi adoptive
transfer model prevented colitis 3
Ulcerative Colitis
(UC)T cells
Not efficacious
in moderate to
severe UC 1
ICOS expression & CD4+ ICOS+ LPMC elevated in inflamed mucosa
compared to non-inflamed or PBMCs 2
ICOS expression on CD4+ LPMC directly correlates with endoscopic
disease activity scores (Matts grade >3)
Immune-Related
Adverse Event ColitisCheckpoint Inhibitor-Mediated
T cells Not TestedCD4+ ICOS+ T cells elevated in lamina propria in checkpoint inhibitor-
mediated colitis from patients treated with anti-CTLA-4 and anti-PD-1 4
Celiac Disease
(CeD)T cells Not Tested
Several studies identified genetic linkage & association of CeD with the
2q33 locus, a region harboring the genes CD28, CTLA-4, and ICOS
(CELIAC3), three important regulators of T cell activity 5,6,7
1 Sandborn 2012: Gastroenterology 143; 2 Sato 2004: Gastroenterology 126; 3 de Jong 2004: Int Immunol 16; 4 Coutzac 2017: J Crohns Colitis 10; 5 Amudsun 2004: Tissue Antigens 64; 6 Gough 2005: Immunological Reviews 204; 7 Haimila 2009: Genes&Immunity 10
0
100000
200000
300000
400000 IFN Response
IFN
- (
pg
/mL
)
1000
2000
3000
4000
5000 IL-6 Response
IL-6
(p
g/m
L)
0
100
200
300
500
1500 IL-12p70 Response
Donor Type
IL-1
6 (
pg
/mL
)
CD UC
0
6000
12000
18000 TNF Response
TN
F-
(p
g/m
L)
DAI: Two-way ANOVA Fc ctrl. vs ALPN-101
Single dose d0 Rpt. dose d0-41 Single dose d14 Rpt. Dose 14-41
p = 0.0002 p < 0.0001 p = 0.0001 p < 0.0001
(D) T Effector Memory (TEM) Cell Suppression
0
1×104
2×104
3×104
4×104
5×104 CD4+ T-cell Recovery
To
tal # C
D4
+ C
ells
0
5×104
1×105
1.5×105
2×105 CD4+ T-cell Recovery
To
tal # C
D4
+ C
ells
0
1×104
2×104
3×104
4×104 #CD4+CD28+ Cells
# C
D4
+C
D28
+ E
ven
ts
0
5×104
1×105
1.5×105 #CD4+CD28+ Cells
# C
D4
+C
D28
+ E
ven
ts
0
1×104
2×104
3×104
4×104 #CD4+ICOS+ Cells
# C
D4
+IC
OS
+ E
ven
ts
0
5×104
1×105
1.5×105
2×105 #CD4+ICOS+ Cells
# C
D4
+IC
OS
+ E
ven
ts
0
50
100
150 IL-6
IL-6
(p
g/m
L)
0
5
10
15
20
25 IL-12p70
IL-1
2p
70
(p
g/m
L)
0
10
20
30
40
50 IFN
IFN (
pg
/mL
)
0
10
20
30
40
50 TNF
TN
F
(p
g/m
L)
0
1×104
2×104
3×104
4×104
5×104 CD4+ TEM Recovery
To
tal # C
D4
+ T
EM
0
5×104
1×105
1.5×105
2×105 CD4+ TEM Recovery
To
tal # C
D4
+ T
EM
0
200
400
600
800CD4+ TREG Recovery
To
tal # C
D4
+ T
RE
G
0
5×103
1×104
1.5×104
2×104 CD4+ TREG Recovery
To
tal # C
D4
+ T
RE
G
(E) Inhibition of Disease-Relevant Cytokines
Blood Mesenteric Lymph Node
0
4
8
12
Colo
n w
gt (m
g)/
length
(m
m)
Colon Weight : Length
0
2
4
6
8
10
Com
bin
ed S
co
re
Combined Histology Score
p <0.0001
p = 0.0003
p <0.0001
p = 0.0016p <0.0001
p <0.0001
No colitis Fc-Control
Single
Dose d0
Delayed
Repeat
Dose
Repeat
Dose
Delayed
Single
Dose d14
ALPN-101 No colitis Fc-Control
Single
Dose d0
Delayed
Repeat
Dose
Repeat
Dose
Delayed
Single
Dose d14
ALPN-101
No
Colitis
Fc
control
ALPN-101 No
Colitis
Fc
controlALPN-101 No
Colitis
Fc
controlALPN-101 No
Colitis
Fc
control
ALPN-101
Rpt. Dose Rpt. Dose Rpt. Dose Rpt. Dose
