ACR ANNUAL MEETING » NOVEMBER 8-13 » ATLANTA, GA, USA

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INTRODUCTION: ALPN-101 is an Fc fusion protein of a human inducible T cell costimulator ligand (ICOSL) variant

immunoglobulin domain (vIgD™) designed to inhibit simultaneously the CD28 and ICOS costimulatory pathways. CD28 and ICOS

each play a role in T cell activation and adaptive immunity which can contribute to autoimmune disease when dysregulated.

ALPN-101 has previously been shown to have potent immunosuppressive activity in various in vitro and in vivo models of disease,

including acute graft versus host disease, inflammatory arthritis, and multiple sclerosis. We report here in vitro assays using

peripheral blood mononuclear cells (PBMC) from healthy donors vs. patients to analyze human T cell and B cell activation and

suppression of antibodies and inflammatory mediators thought to contribute to the pathogenesis of Sjögren’s syndrome (SjS) and

other connective tissue diseases. Additionally, the efficacy of ALPN-101 was confirmed in vivo in mouse immunization models, and

in mouse models of SjS and systemic lupus erythematosus (SLE).

METHODS: Primary cell assays were performed with healthy donor and SjS patient PBMC stimulated with K562 cells expressing

CD80, CD86, ICOSL, and anti-CD3 (OKT3) to evaluate the potency of ALPN-101 to suppress cytokine production and alter gene

expression. The activity of dual pathway inhibition by ALPN-101 was compared to the CD28-only inhibitor abatacept (CTLA4-Ig;

Bristol-Myers Squibb, via Catalent) and to the ICOS pathway inhibitor prezalumab (AMG-557/anti-ICOSL, Creative Biolabs), or a

combination of the two. ALPN-101 was compared to abatacept in vivo in standard mouse immunization models (KLH, sheep RBC),

in a model of SjS involving anti PD-L1 antibody-mediated acceleration of sialadenitis in non-obese diabetic (NOD) mice, and in the

bm12 inducible model of lupus.

RESULTS: Compared to abatacept, prezalumab, or combination abatacept + prezalumab, ALPN-101 demonstrated superior

suppression of pro-inflammatory cytokine (i.e. TNFα, IFNγ, IL-1β, IL-2, IL-6, IL-17A, GM-CSF, etc.) release from stimulated healthy

SjS, or SLE patient PBMCs (Fig. 3). ALPN-101 treatment reduced germinal center (GC) B cells and follicular helper T cells (TFH)

and inhibited antibody production in vivo in mouse immunization models (not shown) and in the bm12 model (Fig. 9), and

suppressed proliferation and antibody production in human B cell/TFH cell co-cultures (Fig. 5). In anti PD-L1-treated NOD mice,

ALPN-101 suppressed sialadenitis, insulitis, blood glucose levels, and autoantibodies with activity often superior to that of

abatacept (Fig. 6-8, and data not shown).

CONCLUSION: The efficacy of dual CD28/ICOS antagonist ALPN-101 is superior to CD28 or ICOS costimulatory pathway

inhibitors, administered individually or in combination, in human in vitro and/or mouse in vivo translational studies. A Phase 1

clinical trial with ALPN-101 in healthy volunteers is ongoing (NCT03748836), and trials in inflammatory diseases are planned.

Stacey R. Dillon, Lawrence S. Evans, Katherine E. Lewis, Susan Bort, Erika Rickel, Jing Yang, Martin F. Wolfson, Kayla Susmilch, Sherri Mudri, Steven D. Levin, Janhavi G. Bhandari, Fariha Ahmed-Qadri, Mark W. Rixon, Jan L. Hillson, Stanford L. Peng, and Kristine M. SwiderekAlpine Immune Sciences, Inc. Seattle, WA

ALPN-101, a First-in-Class Dual ICOS/CD28 Antagonist, Suppresses Key Effector Mechanisms Associated with

Sjögren’s Syndrome and Systemic Lupus Erythematosus

Abstract

Summary and Conclusions

Figure 1: ALPN-101 (ICOSL vlgD-Fc), Generated Using the vIgD™ Directed Evolution

Strategy, Blocks Both CD28 and ICOS T Cell Costimulation Pathways

Figure 3: ALPN-101 Inhibits Cytokine Production from Human Sjögren’s and SLE

Patient PBMC In Vitro More Potently Than Single CD28 or ICOS Pathway Inhibitors

Sponsor: Alpine Immune Sciences, Inc. AlpineImmuneSciences.com @AlpineImmuneSci © 2019 Alpine Immune Sciences. All rights reserved.

Figure 7: ALPN-101 Reduces the Incidence and Severity of Sialadenitis in NOD Mice

Enrolled in the Anti PD-L1 mAb-Induced Model of Sjögren’s Syndrome

Figure 8: ALPN-101 Reduces Serum Autoantibody Titers in the Anti PD-L1 mAb-Induced

NOD Mouse Model of Sjögren’s

(A) Autologous circulating human B and TFH cells isolated from PBMC from healthy donors were mixed with artificial APCs (K562/CD80) and soluble OKT3, and

titrations of the test articles indicated. Cultures were assessed on Day 7 for cellular activation/proliferation and IC50 values are summarized in the table.

(B) In a separate study using a similar protocol, supernatants were collected on Day 8, diluted 1:10, and assayed for human immunoglobulin (Ig) secretion. Data

in (B) are representative of results from 8 donors.

(A) In vitro stimulation of healthy

donor, Sjögren’s syndrome (SjS),

and SLE patient PBMC with

artificial APC [fixed K562

expressing cell surface OKT3 (anti

CD3)/CD80/ CD86/ICOSL)] at a

20:1 ratio. Test articles were added

at 100 nM and supernatants were

collected and assayed for cytokine

concentrations after 48 hr

incubation. (B) IFNγ, IL-6, and

IL-17A levels are shown as

examples, though similar trends

were observed for all cytokines

measured, as summarized in C.

(C) ALPN-101 demonstrated

statistically significant superiority

to prezalumab, abatacept, or a

combination of prezalumab+

abatacept for the majority of

donors and analytes tested.

2416

Figure 2: ALPN-101 Binds CD28 and ICOS and Blocks Binding to Their Ligands,

Impacting B Cell/T Cell Collaboration During Antibody Responses

Figure 6: The Anti-PD-L1 mAb-Induced NOD Mouse Model of Sjögren’s Syndrome

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A mouse model of Sjögren’s syndrome induced in female diabetes-prone NOD/ShiLtJ mice using repeat dosing (on Days 0, 2, 4, and 6) with anti-mouse PD-L1

antibody (based on a modified version of a protocol published by Zhou et al., 20165) was used to evaluate the impact of ALPN-101 and comparator treatments on

the development of sialadenitis and insulitis, the levels of blood glucose, serum autoantibodies, and gene expression in the submandibular gland (SMG).

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Fc Control

ALPN-101

Abatacept

WT ICOSL-Fc

Aba + WT

Naïve

CD28/CTLA-4 inhibition ICOS-only inhibitionCD28+ICOS dual blockade

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Prezalumab

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ICOSL ICOS

[ pM ]

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Prezalumab

ALPN-101

Binding to Targets Blockade of Ligand Binding

• ALPN-101 (ICOSL vIgD-Fc) is a dual CD28 and ICOS T cell co-stimulation pathway inhibitor targeting both naïve

and activated pathogenic T cells, including ICOS+ cells that escape currently available CD28 pathway inhibitors.7

• ALPN-101 inhibits cytokine production in vitro from human Sjögren’s and SLE patient PBMC, more potently than

single CD28 or ICOS pathway inhibitors.

• ALPN-101 affects genes involved in B cell differentiation and suppresses proliferation and antibody responses in

vitro in human B cell-TFH cell co-cultures.

• ALPN-101 suppresses anti-SRBC (and anti-KLH, not shown) antibody responses in vivo in normal mice, and

reduces autoantibody titers in mouse models of Sjögren’s syndrome and SLE.

• ALPN-101 reduces the incidence and severity of sialadenitis and insulitis, and reduces blood glucose levels (Fig. 7

and data not shown), in NOD mice enrolled in the anti PD-L1-induced model of Sjögren’s syndrome.

• ALPN-101 is a novel therapeutic candidate for Sjögren’s syndrome, SLE, and potentially other connective tissue

and/or serious autoimmune diseases. A phase 1 clinical trial with ALPN-101 in healthy volunteers is ongoing

(NCT03748836), and trials in inflammatory diseases are planned.

1. Panneton et al. (2019) Inducible T cell Costimulator: Signaling Mechanisms in T Follicular Helper Cells and Beyond. Immunol Rev. 291:91-103

2. Paulos et al. (2010) The Inducible Costimulator (ICOS) is Critical for the Development of Human TH17 Cells. Sci Transl Med. 2(55):55ra78.

3. Weber et al. (2015) ICOS Maintains the T Follicular Helper Cell Phenotype by Down-Regulating Krüppel-Like Factor 2. J Exp Med. 212(2):217-33.

4. Evans et al. (2019) ALPN-101, a First-in-Class Dual ICOS/CD28 Antagonist, Suppresses Key Effector Mechanisms Underlying Rheumatoid and

Psoriatic Arthritis. Poster #1531, American College of Rheumatology Annual Meeting 2019; Atlanta, GA.

5. Zhou et al. (2016) Endogenous Programmed Death Ligand-1 Restrains the Development of Sjögren’s Syndrome in Non-Obese Diabetic Mice.

Sci Rep. 6: 39105.

6. Klarquist & Janssen (2015) The bm12 Inducible Model of Systemic Lupus Erythematosus (SLE) in C57BL/6 Mice. J Vis Exp. (105): e53319.

7. Dillon et al. (2019) ALPN-101, a Dual ICOS/CD28 Antagonist, Demonstrates Potent and Dose-Dependent Suppression of Graft vs. Host Disease

(GvHD) in a Human/NSG™ Mouse Xenograft Model, with Activity Superior to CD28 or ICOS Single Pathway Antagonists. Poster #426,

Transplantation & Cellular Therapy Meeting of ASBMT and CIBMTR, February 2019, Houston, TX.

ReferencesIC50

17.2 nM

23.3 nM

4.3 nM

Figure 9: ALPN-101 Reduces GC B and TFH Cells and Suppresses Antibody Responses

in Normal Mice and in the bm12 Inducible Mouse Model of SLE

Anti-mouse PD-L1

mAb (0.1 mg), IP

• Dose with anti-mPD-L1 mAb (0.1 mg)• Treat with 0.3 mg Fc control, 0.5 mg ALPN-101,

abatacept, or WT ICOSL-Fc, or 0.5 mg aba + 0.5 mg WT ICOSL-FcNOD/ShiLtJ female,

6 wks of age

Day 10

Day 10 Harvest:• SMG (histology, RNA-Seq)

• Pancreas (histology)

• Blood/serum (glucose, PK)

ALPN-101 (0.5 mg), IP

Day 0 Day 2 Day 4 Day 6

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Donor Type

IFN (

pg

/mL

)

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Donor Type

IL-1

7A

(p

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Fc control

Prezalumab

Abatacept

Abatacept+Prezalumab

ALPN-101

Anti ICOSL mAb

(prezalumab)

ICOS only

Abatacept

(CTLA4-Fc)

CD28 only

ALPN-101

Monotherapy

ICOS+CD28

Systemic Lupus

Erythematosus

Sjögren’s

SyndromeHealthy

Donors

% Inhibition

vs. Fc

(B) Examples of cytokine responses in the PBMC stimulation assay

(C) Summary of cytokine responses in the PBMC stimulation assay

(A) Diagram of the PBMC stimulation assay Artificial APCK562/OKT3/CD80/CD86/ICOSL

T cell

CD28

ICOS

CD80/86

ICOSL

OKT ScFv

Target(s)Antagonists

ICOSLPrezalumab

CD80/

CD86

Abatacept

Belatacept

ALPN-101CD28

ICOS

(A) BALB/c mice were immunized with SRBC on Day

0 (D0). Half the mice were dosed on D-1 & D7, and

half on D14 & D21 with 1.5 mg ALPN-101 or

abatacept, or a molar-matched amount of Fc control.

The second group was boosted with SRBC on D15

and all mice were euthanized 1 wk after their 2nd dose

(on D14 and D28, respectively). Serum anti-SRBC

antibodies on D14 and D28 were measured via flow

cytometry by detecting binding to SRBC using

isotype-specific anti-mouse Ig Abs. p values were

determined using a ordinary one-way ANOVA with

Tukey's multiple comparisons test.

(B) Spleens were harvested on D14 from the first

cohort of mice (treated on D-1 & D7) and analyzed by

flow cytometry.

(C) Per a published protocol6, splenocyte suspensions

from female I-Abm12B6(C)-H2-Ab1bm12/KhEgJ (‘bm12’)

mice were injected IP into an equivalent number (1:1)

of 9 wk old female C57BL/6NJ recipient mice on Day

0. H2-Ab1bm12 differs from H2-Ab1b by 3 amino acids

in the β-chain of the I-A molecule. Alloactivation of

donor CD4+ T cells by recipient APC leads to a

cGvHD disease with symptoms resembling SLE.

Starting on Day 0, recipient mice were treated 2x/wk

for 11 wks with 400 µg ALPN-101 or abatacept, or a

molar-matched amount (250 µg) of Fc control protein.

At study termination on Day 82, GC B cells and TFH

cells were enumerated by flow cytometry. Anti-double

stranded (ds) DNA IgG titers were measured on D14

using the Mouse Anti-dsDNA IgG Antibody Assay kit

from Chondrex (Redmond WA).

Patient or healthy donor peripheral blood mononuclear

cells (PBMC) were stimulated with fixed artificial APCs

(shown at right) for 48 hrs in the presence of ALPN-101

(100nM) or comparators:

o Prezalumab (anti ICOSL mAb)

o Abatacept alone (CTLA4-Ig)

o Abatacept + prezalumab

APC Antigen

CD28+

ALPN-101

Inhibition of T cellactivation

Inhibition of B cellactivation and Ab production

ALPN-101

ICOS+

ActivatedTFH cell

ExistingTFH cell

ICOS+

Memory B cells

Antibody-secreting

plasma cells

NaïveT cell

B cell

o Primary immune responses depend heavily upon the CD28 pathway

o Secondary immune responses (e.g. memory, effector) can be CD28-independent.

o ICOS is upregulated upon T-cell activation and is the most closely related protein to CD28, yet plays a non-redundant function in many immune responses, such as follicular helper T cell (TFH) differentiation and function.1,3

o ICOS+ T cells escape treatment with drugs only blocking the CD28/CTLA-4 pathway, like abatacept.

Our Thesis: Blockade of both CD28 and ICOS pathways by ALPN-101 should provide superior efficacy to therapeutics which only interfere with a single pathway

ICOS-L+

ICOS+

CD80/CD86+

Figure 5: ALPN-101 Suppresses Proliferation and Antibody Responses in Human B-TFH

Cell Co-Cultures and Affects Expression of Genes Involved in B-T Cell Collaboration

% Incidence of Sialadenitis

0.0

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0.4

Serum anti Ro-52

OD

at

1:3

12

.5 d

ilu

tio

n vs ALPN-101:

*** p=0.0002

**** p<0.0001

********

***

0.0

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0.4

Serum anti La

OD

at

1:3

12.5

dil

uti

on ***

**

^

vs ALPN-101:

^ p=0.0146

** p=0.0026

*** p=0.0002

Fc Control

ALPN-101

Abatacept

WT ICOSL-Fc

Aba + WT ICOSL-Fc

Naïve

Figure 4: ALPN-101 Suppresses Expression of Many Genes Associated with

SjS/SLE Mechanisms and Phenotypes Individual SMG Histology Scores

• CCL20

• CD40LG• CSF2

• ICOS

• ID2

• IFNG• IGSF3

• IL17A• IL1R1

• IL2

• IL21• IL24

• IL3

• IL31

• IL6• IL9

• LIF

• NFKBID

• PDCD1

• TNF

• BATF3

• CTLA4

• FLT1

• FOSL1

• IL13

• IL17F• IL1R2

• IL23R• IL4

• IL5

• VEGFA

• CCL17

• CXCR5• GATA3

• IL12RB2

• IL18R1

• IL18RAP

• KITLG

• LTA

• PVR

• SOCS3

• TFRC

• TNFRSF8

• RNA was isolated from patient PBMC

samples generated in the assay

described in Fig. 3

• Genes significantly down-regulated in

response to ALPN-101 across SjS, SLE,

and psoriatic arthritis (PsA) stimulated

patient populations suggest ALPN-101

can impact pathogenic genes and/or

pathways implicated in many

autoimmune diseases.

Anti Ro-52 and anti La Abs were detected by

ELISA (Signosis, Santa Clara, CA) in diluted

serum collected on Day 10 from mice in the

SjS study described in Figs 6-7. Statistical

differences noted between the ALPN-101

group and comparator treatments were

determined using an uncorrected non-

parametric Dunn’s test.

See also: Poster #1531. Evans et al. (2019) ALPN-101, a First-in-Class Dual ICOS/CD28

Antagonist, Suppresses Key Effector Mechanisms Underlying Rheumatoid and Psoriatic

Arthritis. ACR Annual Meeting; November 11, 2019.4

(A) Human T and B Cell Proliferation (B) Human Immunoglobulin Production

BetterWorse

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CD4+ T cells: CD40L+ Cell Count

[ pM ]

Cell #

ICOSWT ICOSL IgV

Aba + WT ICOSL-Fc

1 µm

Representative H&E Images of SMG

(C) RNA expression analysis of key genes involved in B-T cell collaboration (C) RNA isolated from a B/TFH co-culture similar to the one

described in A was analyzed for relative gene expression levels

by next generation sequencing. Expression values in RPKM are

plotted on a relative scale from 0 to 1 for each gene between

treatment groups, with degree of red reflecting higher

expression and degree of blue representing lower values for

each gene. The list of genes shown was sorted by level of

inhibition by ALPN-101 (least to most inhibited, from L→R).

Statistical significance determined using the Kruskal-Wallis test: * p < 0.05, ** p < 0.01,

**** p < 0.0001. The naïve group was not powered for statistical comparisons.

IC50

1.0 nM

5.4 nM

2.5 nM

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IgG

an

ti-d

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NA

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L)

Serum anti-dsDNA (IgG)

Fc Control

ALPN-101

Abatacept

Naïve BL/6

-1×105

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***** ** ** ****ALPN-101 inhibits both CD28 and

ICOS-mediated costimulation.

Comparators such as abatacept /

belatacept (CTLA4-Ig, BMS) or FR104

(anti CD28 Fab, OSE

Immunotherapeutics) inhibit only the

CD28/CTLA-4 pathway, while

prezalumab (anti ICOSL mAb, Amgen)

inhibits only ICOS costimulation.

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.1

ICOS signaling is crucial for the

development of human TH17 cells2 and

the function & maintenance of follicular

helper T cells.3 ALPN-101 may thus be

uniquely suited to treat diseases

driven by these pathogenic cell types.

0.001 0.01 0.1 1 10 100 10000

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[Test Article], nM

[Hu

Ig

G1],

ng

/ml

Mean Fc control

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[Test Article], nM

[Hu

Ig

G3],

ng

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Mean Fc control

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[Test Article], nM

[Hu

Ig

M],

ng

/ml

Mean Fc control

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[Test Article], nM

[Hu

Ig

A],

ng

/ml

Mean Fc control

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

D14 D280

500

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Day Post-Immunization

MF

I

1:136 Diln1:45 Diln

**

D14 D280

5000

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MF

I

1:409 Diln1:45 Diln

******** **

***

(A) Serum Ig titers in BALB/c mice immunized with sheep red blood cells (SRBC)

SRBC Day 0

Treated on D-1 and D7

Sac D14

GC B = B220+CD19 + GL7 + CD95 +

TFH = CD4 + CD45 + CD3 + PD1 + CD185 +

(B) Numbers of splenic GC B and TFH in BALB/c mice 14 days after immunization with SRBC

0

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4+ T

FH c

ell #

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03) # CD4+ TFH cells/spleen

(C) Numbers of splenic GC B and TFH and serum anti-dsDNA titers in the bm12 inducible model of SLE

D14 D280

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MF

I

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*******

****

D14 D280

10000

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50000

Day Post-Immunization

MF

I

1:45 Diln 1:45 Diln

******

***

IgM anti-SRBC IgG1 anti-SRBC IgG2a anti-SRBC IgG2b anti-SRBC

Statistical significance was determined for GC B and TFH using an unpaired t-test, and for anti dsDNA concentrations using an uncorrected Fisher’s least significant differences test; *p<0.05, **p<0.01, and ****p<0.0001.

* p<0.05

** p<0.005

*** p<0.001

**** p<0.0001

WT ICOSL-FcAbataceptALPN-101Fc control