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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