Novel Variant Ig Domain (vIgD) Proteins Generated Via Directed Evolution of IgSFDomains Have Therapeutic Efficacy in Animal Models of Graft Versus Host Disease

Steven D. Levin, Lawrence S. Evans, Erika Rickel, Katherine E. Lewis, Rebecca P. Wu, Martin F. Wolfson, Stacey R. Dillon,

Michael G. Kornacker, Ryan Swanson, and Stanford L. Peng

Alpine Immune Sciences, Inc., Seattle, Washington, USA

Abstract

Figure 1: Directed Evolution Strategy Selecting Protein Variants with Altered Binding Properties

Figure 7: ICOSL vIgD-Fcs Protect from GvHD in vivo

Summary and Conclusions

The immunoglobulin superfamily (IgSF) is a large, diverse family of proteins extensively targeted for treatment of cancers and autoimmune diseases. Most of

the therapeutic strategies targeting this family have focused on high affinity antibodies binding a single receptor. Wild-type IgSF receptors typically exhibit low

affinities for their counter-structures, limiting their utility in therapeutic modulation of immune responses. We have developed a novel variant Ig domain™

(vIgD™) directed evolution platform to affinity mature human IgSF extracellular domains. In this platform, libraries of mutagenized IgSF domains are selected for

altered affinity to specific recombinant protein counterstructures. Fc fusion proteins incorporating the resulting engineered IgSF domains are then tested in vitro

for their ability to either agonize or antagonize T cell responses.

We successfully engineered the IgSF protein ICOSL, which binds to the costimulatory molecule ICOS on T cells and triggers T cell activation. We used our

directed evolution platform to engineer a first-in-class single vIgD domain with higher affinity for ICOS and high affinity binding to the costimulatory molecule

CD28, which ICOSL does not normally bind. Following the creation of dual ICOS/CD28 antagonism, the resulting domains were used to generate individual

constructs encoding Fc-fusion proteins. These enhanced ICOS/CD28 dual antagonist Fc-fusion proteins were produced in HEK-293 cells and purified proteins

were then tested in a FACS based binding assay on cells transfected with each costimulatory receptor showing up to a 2-3-fold increase in ICOS binding and

high affinity binding to CD28. Various ICOS/CD28 dual antagonist Fc-fusion proteins were then tested in soluble format over a range of concentrations for

functional activity in mixed lymphocyte reaction cultures (MLR) where allogeneic dendritic cells (DC) were used to stimulate purified human T cells. Most

variants showed significant ability to inhibit the MLR response as read out measuring proliferation and cytokine production. The potency of the Fc-fusion proteins

varied somewhat depending on their relative affinities for CD28 and ICOS, with all being superior to wild type ICOSL protein and many showing superiority to the

CD28 blocking reagent belatacept (CTLA4-Fc). The level of inhibition correlated well with binding of the Fc-fusion proteins to the T cells in culture.

To assess the function of the ICOS/CD28 dual antagonists in vivo, ICOSL vIgD-Fc proteins were tested in the human PBMC-NSG™ GVHD mouse model. This

model monitors graft versus host disease generated by xenogeneic responses of T cells in human PBMCs transferred into NSG™ mice. Administration of the dual

ICOS/CD28 antagonist Fc-fusion proteins significantly protected mice from the effects of xenogeneic T cell activation in vivo with treated animals showing

dramatically enhanced survival and greatly reduced disease scores. The level of protection correlated with the potency of the molecules in the in vitro MLR

assay with less potent molecules in MLR assays failing to protect while more potent molecules protected well. Belatacept was also effective in protecting

animals from GVHD, but not as effective as the ICOS/CD28 dual antagonists produced by the vIgD platform. Collectively, these data demonstrate the vIgD

directed evolution platform can generate IgSF proteins with altered affinities and improved binding properties. Variants engineered for desired immunological

properties can effectively perturb T cell costimulation and affect T cell responses both in vitro and in vivo with the in vitro potency of the proteins correlating to in

vivo potency. The dual ICOS/CD28 antagonists produced with the vIgD platform can be used therapeutically in disease settings where attenuation of T cell

responses is beneficial, including GvHD and potentially other autoimmune diseases. Phase I enabling studies are in progress.

0 5 10 15 20 25 30 35 40 45 500

25

50

75

100

HuPBMC-->NSG GVHD

Day

% S

urv

ival

Saline

WT ICOSL

A2229

A2237

A2231

belatacept

last dose(d. 37)

A184

0 5 10 15 20 25 30 35 40 45 500

2

4

6

8

HuPBMC-->NSG GVHD

Day

Mean

DA

I S

co

re

Saline

WT ICOSL

A2229

A2237

A2231

belatacept

(Survival)

(0/9)

(9/9)

(0/9)

(7/9)

(6/9)

(0/9)

last dose(d. 37)

A184 (8/9)

Figure 7. ICOSL vIgD-Fc proteins suppress immune responses in vivo. An acute model of graft-versus-host-disease (GvHD) was performed by adoptively transferring human PBMC into immunodeficient NSG mice (n=9/group). High affinity ICOSL vIgD-Fc proteins significantly (a) prolonged survival and (b) reduced mean disease activity index (DAI; a score of overall health and weight loss). Administration of high affinity ICOSL

vIgD-Fc proteins protected from effects of GVHD at levels comparable to or better than belatacept, but wild-type ICOSL-Fc or a lower affinity ICOSL vIgD-Fc fusion protein (A2231) were not effective in protecting from GVHD in this model. (a) By log-rank test, belatacept and high affinity ICOSL-Fc proteins significantly prolonged survival as compared to saline and WT ICOSL-Fc (p < 0.001); ICOSL vIgD-Fc A2237 prolongs survival as compared to belatacept (p = 0.065). (b) By 2-way repeated-measures ANOVA, belatacept and high affinity ICOSL vIgD-Fc proteins significantly reduce DAI scores as compared to saline and wild-type ICOSL-Fc (p <0.001); ICOSL-Fc A2229 and A2237 reduce DAI scores as compared to belatacept (p = 0.053 and p = 0.035, respectively).

• A variant Ig domain (vIgD) platform has been developed to generate novel

immunomodulatory IgSF-based protein therapeutics with increased affinity and multiplicity of

ligand binding, translating into superior preclinical efficacy in vitro and in vivo

• ICOSL vIgD-Fcs demonstrate novel, high affinity binding to CD28 and ICOS and can inhibit

these costimulatory pathways

• ICOSL vIgD-Fcs demonstrate superior efficacy to belatacept (CTLA4-Ig) in vitro in human MLR

assays including inhibition of T cell proliferation and cytokine production, including many of the

cytokines induced in a GVHD response

• ICOSL vIgD-Fcs protected better than belatacept in a humanized GVHD in vivo model

• The vIgD therapeutic platform has broad potential to enhance the activity of biologics in

treatment of human disorders driven or subject to modulation by IgSF proteins, including

autoimmunity, cancer and infectious diseases

• Preclinical development of ICOSL vIgD-Fc (ALPN-101) is underway to support clinical studies

Figure 6: ICOSL vIgD-Fc Proteins Suppress T Cell Responses in vitro

Figure 6. Soluble, engineered ICOSL vIgD-Fc proteins effectively attenuate T cell responses in a human mixed lymphocyte reaction

(MLR). Monocyte derived dendritic cells from one donor were used to allogeneically stimulate T cells from another donor.

Responses were measured by assessing (a) CD4 T cell proliferation, (b) CD8 T cell proliferation, (c) IFN-γ levels in culture

supernatants, and (d-e) the percentage of CD4+ T cells that stained for intracellular IL-21 (d) or IL-4 (e). Proliferation results use

CFSE dilution to show the percentage of divided cells versus protein concentration. IFN-γ levels are shown as pg/ml versus protein

concentration. Intracellular cytokine staining is reported as (d) the MFI of CD4+ IFN-γ + T cells or (e) the percentage of CD8+ T cells

that stained for TNF. (f) IC50 values (pM) were calculated based on IFN-γ release data in (c) using GraphPad Prism. ND = could

not be determined. Results shown are representative of at least three separate experiments performed with each protein.

Disease Activity IndexSurvival

Figure 4: CD28 and ICOS Costimulation in Graft Versus Host Disease

Figure 5: ICOSL vIgD-Fc, an ICOS/CD28 Dual Antagonist for Autoimmunity/Inflammation

Figure 8: ICOSL-vIgD Prevent T Cell Activation in vivo and Preserve Naïve T Cell Phenotype

a. b.

Figure 5. Schematic

showing proposed

mechanism of action for

ICOSL vIgD-Fc with its

capacity to inhibit both

the CD28 and ICOS T

cell costimulation

pathways

Bead and flow cytometric selection

vIgD

Fc-fusion protein

generation

Counter-structure binding

Functional assays

IgSF ECD Yeast Display Libraries

IgSF protein

Therapeutic protein

Limited counter-structures

with low/moderate affinity

Random or

targeted

mutagenesis of

ICOSL ECDParental ICOSL

binds ICOS

Selections:

rICOS and rCD28

Binding assays

• Flow cytometry

• Octet (affinity)

MLR

• Proliferation

• IFNγ

production

Screen yeast outputs

for improved binding

to rICOS and rCD28

Sequence yeast

outputs and identify

unique variant hits

Transient 293 or CHO

production followed

by Protein A

purification

Tailored counter-structures

with improved/high affinity

Figure 2: Simultaneous Affinity Maturation of ICOSL Towards Two Receptors

Dual-Ligand Affinity Maturation with a Single,

Randomly-Mutated ICOSL vIgD Library

Yeast were transformed with a single, randomly-

mutated vIgD library and affinity matured by

selection with CD28 and ICOS. Individual receptor

binding to bulk yeast populations are shown,

including two rounds of flow cytometric selection.

Improvements to both ligands were noted after 1st

selection (F1), and were further improved after 2nd

selection (F2). MFI, mean fluorescence intensity.

-1 0 1 20

200

400

600

800

CD28 Binding

CD28 Fc log[ nM ]

MF

I

WT ICOSL

F1

F2

-2 -1 0 1 2

200

600

1000

1400

ICOS Binding

ICOS Fc log[ nM ]

MF

I

WT ICOSL

F1

F2

Figure 3: vIgDs can be Formatted Specifically for Various Therapeutic Applications

vIgDs from Alpine’s directed

evolution platform may have

multiple therapeutic formats

• Fusion proteins (Fc or

mAb) with antagonistic or

tumor-localizing agonistic

activity

• Cell-displayed (TIP)™ or

secreted (SIP)™ versions

for enhancement of

adoptive cellular

therapies

d. MFI IFNg CD4+ T Cells e. % TNF+ CD8+ T Cells

a. CD4 T Cell Proliferation b. CD8 T Cell Proliferation c. IFNg Release

Protein IC50

No Stim ND

No Protein ND

Fc Control ND

WT ICOSL-Fc ND

Belatacept 8265

A184 ICOSL vIgD-Fc 113

A2229 ICOSL vIgD-Fc 80

A2231 ICOSL vIgD-Fc 682

A2237 ICOSL vIgD-Fc 75

f. Calculated IC50

Salin

e

ICO

SL vIg

D-F

c

0.0

0.1

0.2

0.3

0.4

0.5

Hu

man

/Mo

use C

ell R

ati

o

p=0.0025

Salin

e

ICOSL v

IgD-F

c

0

500

1000

1500

2000

2500

Cell C

ou

nt

p=0.0101

Salin

e

ICOSL v

IgD-F

c

0

1000

2000

3000

Cell C

ou

nt

p=0.0021

a. Human:mouse cell ratios b. CD4+ T Cell Numbers c. CD8+ T Cell Numbers Figure 8. T cells from blood samples taken on day 6 from GvHD mice either treated with saline or A2337 ICOSL vIgD-Fc were stained for human CD4, CD8, CD62L and CD45RA and mouse CD45 to evaluate effects of treatment on T cell expansion and memory cell development. Treatment with ICOSL vIgD-Fc reduced (a) the ratio of human cells to mouse cells and (b) human CD4+ and (c) CD8+ T cell numbers in the blood. (d) The distribution of naïve and memory cell populations were also assessed

based on expression of CD45RA and CD62L using the gating strategy shown. Treatment with ICOSL vIgD-Fc increased the percentage of cells retaining a naïve phenotype and reduced the number of cells with a central memory profile (Tcm). p values based on Student’s t-test.

Salin

e

A22

37 IC

OSL v

IgD-F

c

25

30

35

40

% P

osit

ive

p=0.0148

Salin

e

A22

37 IC

OSL v

IgD-F

c

25

30

35

40

45

50

% P

osit

ive

p<0.0001

d. Phenotypes of human CD4+ T cells

Central MemoryNaïve

CD45RA

CD

62

L

Saline TreatedA2237 ICOSL

vIgD-Fc Treated

APC T cell

ICOSL vIgD

CD80/CD86

CD28

ICOS

ICOSL

CD80/CD86 CD28

ICOSICOSL

Activating Blocking

Fc

ICOSL

10 100 1000 100000

1000

2000

3000

[Protein] pM

MF

I

0 0.1 1 10 100 1000 1000015

20

25

30

[Protein] pM

% P

os

itiv

e

No Stim

No Protein

Fc Control

Belatacept

WT ICOSL-Fc

A2229 ICOSL vIgD-Fc

A2231 ICOSL vIgD-Fc

A2237 ICOSL vIgD-Fc

Modern therapies targeting the immune synapse

CAR-T (TIP)Autoimmune

ICOSL ECD

Fc

vIgD ICOSL

Oncology

mAb

CostimulatoryAgonist

CostimulatoryV-mAb

Fc

Target 1

Target 2

vIgD Multi-Checkpoint

AntagonistCostimulatoryAgonism

Fc

CheckpointInhibition

Multi-Functional vIgD

Web: www.alpineimmunesciences.com Twitter: @AlpineImmuneSci

1 10 100 1000 10000

0

200

400

600

800

1000

1200

[Protein] pM

IFNg (

pg

/ml)

Fc Control

WT ICOSL

A184

A2229

A2231

A2237

No Protein

No Stim

belatacept

A3269

1 10 100 1000 100000

10

20

30

[Protein] pM

% D

ivid

ed

1 10 100 1000 100000

5

10

15

20

[Protein] pM

% D

ivid

ed

Figure 4. Schematic showing contributions of CD28 and ICOS costimulation to development of GvHD. CD28 and ICOS costimulatory signals drive differentiation of alloreactive T cells in CD4+ or CD8+ effector T cells contributing to multi-organ pathogenesis