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The Variant Immunoglobulin Domain (vIgD) platform• Directed evolution: yeast maturation of individual IgSF domains
• Selection performed by flow cytometry
• Screening of mammalian cell-expressed domains performed via
binding and functional assays
• Iterative process yields unique vIgDs evolved to modulate multiple
counter-receptors
Novel immunomodulatory proteins generated via directed
evolution of variant IgSF domainsKatherine E. Lewis, Lawrence S. Evans, Steven D. Levin, Erika Rickel, Martin F. Wolfson, Susan Bort, Stacey R. Dillon, Sherri Mudri, Michael G.
Kornacker, Ryan M. Swanson, Stanford L. Peng
Alpine Immune Sciences, Inc., 201 Elliott Avenue West, Seattle, Washington USA
Abstract
The immunoglobulin superfamily (IgSF) is a large, diverse family of proteins expressed
on immune cells 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. Moreover, 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™) platform using directed
evolution and yeast display to affinity mature human IgSF extracellular domains. In this
platform, libraries of mutagenized IgSF domains are selected for enhanced or altered
affinity to specific recombinant proteins. Fc fusion proteins incorporating these evolved
immunomodulatory IgSF domains are then tested in vitro for their ability to either
agonize or antagonize T cell responses.
Multiple novel vIgD™ fusion proteins have been generated which significantly
attenuate or accentuate T cell activation in vitro as assessed by proliferation and
cytokine production. Lead molecules also exhibited in vivo efficacy in the human
PBMC-NSG™ GVHD mouse model. Efficacy in vitro and in vivo was superior to wild-type
IgSF domains due to the induced alterations in affinity for cognate ligand and through
specifically-directed changes in their ability to bind additional counter-structures.
Our results demonstrate that vIgDs™ evolved to acquire unique biochemical
properties significantly enhance therapeutic utility as immunomodulatory agents. This
vIgD™ therapeutic platform has broad potential to enhance the activity of biologics in
treatment of autoimmune diseases, cancer, and other disorders.
The Immunoglobulin Superfamily (IgSF)
The vIgD Platform
vIgDs may be used in multiple therapeutic formats• Fusion proteins (Fc or mAb) with inhibitory and/or tumor-localizing agonistic activity
• Cell-displayed for enhancement of adoptive therapies
IgV/IgC Yeast Display
Flow Cytometric Selection
Fc Fusion Protein
Generation
Counter-structure Binding
Functional Assays
IgSF Protein
•Limited
counterstructure(s)
•Low/modest affinity
vIgD
•Multiple and/or
tailored counter-
structure(s)
•Improved/high affinity
Oncology
Immunology and
Autoimmunity
• The immunoglobulin superfamily (IgSF):• Is the largest protein superfamily
• Includes cell surface and soluble members/forms involved in cellular
recognition, binding, and adhesion
• Each IgSF member consists of at least one Ig domain of 70-110 aa each
• Variable (IgV), constant (IgC1, IgC2), or intermediate (IgI)
• Has been most well studied in immunology and immuno-oncology, but
members are also relevant to other therapy areas, including
endocrinology and neuroscience
• Key examples of IgSF that play critical roles in immunoregulation :• 1⁰ immune cell receptors: Igs, TCRs, KIRs, LILRs (Fig. 1)
• Co-receptors: CD4, CD8, CD19, MHC, b2m
• Costimulatory and checkpoint molecules: CD28, CD80, CD86, CTLA-4,
PD-1, LAG-3, TIM-3, BTLA
• CAMs: NCAMs, CD2 family
• Cytokine receptors: IL-1R, CSF-1R
• Growth factor receptors: PDGF-R, c-kit
Figure 1. Model of the Immunological Synapse Including PD-1Proc Natl Acad Sci USA 105:10483, 2008
Poster W.83
Conclusions and Summary
• A variant Ig domain (vIgD) platform has been developed to generate a novel dual
immunomodulatory IgSF-based biologic with higher affinity for and increased multiplicity of
ligand binding, translating into superior preclinical efficacy in vitro & in vivo in these analyses.
• ICOSL vIgDs repeatedly demonstrate superior efficacy to belatacept in these analyses for
inhibition of cytokine secretion, including IL2, IFN-γ, and several TH2 cytokines, in MLR assays.
Cellular proliferation and intracellular cytokine staining are also reduced to a greater extent.
• 1st gen ICOSL vIgDs are as effective as approved CTLA-4 based therapeutics (abatacept and
belatacept) in these DTH and humanized GVHD in vivo models. 2nd gen ICOSL vIgD-Fc
molecules are currently in a follow-up preclinical GVHD study with data to be reported in a
future scientific forum.
• ICOSL vIgDs domains may provide novel therapeutics for the treatment of multiple
inflammatory conditions; preclinical development is underway to support clinical trials.
• Development of ICOSL vIgDs for immuno-oncology applications has also been initiated, with
potentially promising data for the various therapeutic formats generated to date.
Tailored High-Affinity Dual CD28/ICOS Binding of
ICOSL vIgD Domains
Figure 4. Structure of vIgD Fc Fusion Proteins with Improved Binding to Counter Receptors. Yeast
outputs were batch cloned into an Fc expression vector, inserts sequenced, and unique clones of
interest chosen for transient expression in Expi293 cells (left). HEK293 cells were transiently transfected with three distinct IgSF receptors. Cells were stained with titrated WT or mutant vIgD hits. Binding
was detected with PE-conjugated anti-human Fc(each binding curve). MFI, mean fluorescence intensity.
Table 1. Improved Ligand Affinity of vIgD-Fc Proteins. Example dissociation constant (KD) determinations on recombinant ICOSL
vIgD-Fc proteins using 3 counter receptors on a ForteBio Octet. FI, fold increase in affinity vs. wild-type (WT). Note: this is an
avidity-driven system due to the bivalent nature of both the receptors and the vIgDs.
1 2 3 4
0
3 0 0 0 0
6 0 0 0 0
9 0 0 0 0
1 2 0 0 0 0
1 5 0 0 0 0
C D 2 8 B in d in g
v Ig D lo g [ p M ]
MF
I
1 2 3 4
0
7 5 0 0 0
1 5 0 0 0 0
2 2 5 0 0 0
3 0 0 0 0 0
C T L A -4 B in d in g
v Ig D lo g [ p M ]
MF
I
1 2 3 4
0
3 0 0 0 0
6 0 0 0 0
9 0 0 0 0
1 2 0 0 0 0
IC O S B in d in g
v Ig D lo g [ p M ]
MF
I
- - - W T IC O S L
1st
G e n v Ig D s
2n d
G e n v Ig D s
3r d
G e n v ig D s
KD [pM] FI KD [pM] FI KD [pM] FI
WT ICOSL 13880 - 77120 - 883 -
525 26 678 114 332 2.7
1163 12 1522 51 338 2.6
783 18 833 93 769 1.1
436 32 647 119 382 2.3
896 15 1466 53 1294 0.7
447 31 645 120 492 1.8
401 35 592 130 373 2.4
390 36 576 134 472 1.9
293 47 536 144 420 2.1
368 38 719 107 369 2.4
1042 13 2518 31 337 2.6
503 28 1059 73 543 1.6
553 25 648 119 362 2.4
563 25 1066 72 477 1.9
366 38 864 89 477 1.9
969 14 1122 69 340 2.6
1947 7 2812 27 371 2.4
910 15 1200 64 311 2.8
3rd Gen
ICOSL
vIgDs
Sensor Load
CD28 CTLA-4 ICOSSample ID
1st Gen
ICOSL
vIgDs
2nd Gen
ICOSL
vIgDs
ICOSL ECD
IgV and IgC
Domains
Fc
Enhanced in vitro Activity of Inhibitory vIgD
Above that of Belatacept (CTLA-4 Ig)
Figure 5. Inhibitory Activity of Antagonistic
ICOSL vIgDs in Mixed Lymphocyte Responses.
vIgD-Fc fusion proteins were evaluated for
their ability to attenuate cytokine levels and T
cell proliferation in a mixed lymphocyte
reaction (MLR). Purified T cells were CFSE-
labeled and incubated with allogeneic
monocyte-derived dendritic cells for 4 days in
the presence or absence of various Fc-fusion
proteins and belatacept (CTLA-4 Ig).
Supernatants were collected and cytokine
concentrations determined by cytokine
bead array (top 2 panels). Proliferation was
determined by quantifying the percentage
of cells that had diluted the CFSE. Effects on
CD8+ T cells and CD4+ T cells are presented
(center panel). Intracellular cytokine staining
was performed on cells stimulated with
PMA/ionomycin in the presence of brefeldin
A and monensin (bottom panel).
0 .0 1 0 .1 1 1 0 1 0 0
1 0 0
2 0 0
3 0 0
4 0 0
5 0 0
IL -1 3
F c P ro te in [ n M ]
Cy
tok
ine
[ p
g/m
L]
0 .0 1 0 .1 1 1 0 1 0 0
0
5 0 0
1 0 0 0
1 5 0 0
IL -2
Cy
tok
ine
[ p
g/m
L]
0 .0 1 0 .1 1 1 0 1 0 0
1 0 0 0
3 5 0 0
6 0 0 0
8 5 0 0
1 1 0 0 0
IF N -
Cy
tok
ine
[ p
g/m
L]
0 .0 1 0 .1 1 1 0 1 0 0
0
1 0 0
2 0 0
3 0 0
IL -6
Cy
tok
ine
[ p
g/m
L]
0 .0 1 0 .1 1 1 0 1 0 0
5 0
1 5 0
2 5 0
IL -9
F c P ro te in [ n M ]
Cy
tok
ine
[ p
g/m
L]
0 .0 1 0 .1 1 1 0 1 0 0
2 5
7 5
1 2 5
1 7 5
2 2 5
IL -5
F c p ro te in [ n M ]
Cy
tok
ine
[ p
g/m
L]
0 .0 1 0 .1 1 1 0 1 0 0
0
5 0
1 0 0
1 5 0
T N F -
Cy
tok
ine
[ p
g/m
L]
0 .0 1 0 .1 1 1 0 1 0 0
5 0
1 5 0
2 5 0
3 5 0
4 5 0
5 5 0
IL -2 2
F c P ro te in [ n M ]
Cy
tok
ine
[ p
g/m
L]
0 .0 1 0 .1 1 1 0 1 0 0
1 5
2 5
3 5
4 5
C D 8 + T -c e lls
% D
ivid
ed
Ce
lls
0 .0 1 0 .1 1 1 0 1 0 0
1 5
2 5
3 5
C D 4 + T -c e lls
% D
ivid
ed
Ce
lls
0 .0 1 0 .1 1 1 0 1 0 0
5
1 0
1 5
2 0
2 5
IL -2 1 + C D 4 +
P ro te in [ n M ]
% P
os
itiv
e
0 .0 1 0 .1 1 1 0 1 0 0
1 5
2 0
2 5
3 0
IF N - + C D 4 +
P ro te in [ n M ]
% P
os
itiv
e
Enhanced in vivo Activity With Inhibitory vIgD
GVHD % Body Weight Loss(Mean + SEM)
PB
S
Ab
ata
cep
t
0
5
1 0
1 5
2 0
Ch
an
ge
in
Ea
r T
hic
kn
es
s
(in
. x
10
-2
)
2n d
G e n IC O S L
v Ig D s
p < 0 .0 0 1 v s . P B S b y 1 -w a y A N O V A
Figure 7. Efficacy of 1st Generation ICOSL vIgD Fc in Graft-Versus-Host Disease (GVHD) Competitive vs. Belatacept. Human PBMC-engrafted NSG™ mice were treated with saline, belatacept (CTLA-4 Ig), or wild-type (WT) or 1st generation ICOSL vIgD-Fc fusion proteins (n=10/group). Mice were
assessed for body weight (BW) loss and a disease activity score (overall health and activity, skin and hair changes, and BW loss) (right panel). 2nd generation ICOSL vIgD-Fc molecules are currently being evaluated in a follow-up huPBMC-NSG GVHD model to be reported at a later date.Model performed at The Jackson Laboratory.
Figure 6. Greater Activity of ICOSL vIgD-Fc than Abatacept in Mouse Delayed
Type Hypersensitivity (DTH) Model. Female BALB/cN mice were primed with ovalbumin emulsified in adjuvant (Sigma) at the base of the tail. On D7,
mice were dosed with PBS, abatacept (CTLA-4 Ig), or ICOSL vIgD-Fc fusion
proteins. Baseline ear measurements were taken, followed by 2⁰ challenge
with intradermal injection of ova.
Change in ear thickness determined 24 hours later.
Delayed-Type Hypersensitivity (Ear Edema) Model
Dual Roles of ICOS and CD28 in Costimulation
Figure 3. ICOSL and Potential Therapeutic Applications. Inducible
costimulator ligand (ICOSL) provides a positive secondary signal to
T cells upon binding to its high affinity receptor, ICOS. ICOSL also
has a natural, low affinity for a second costimulator, CD28 (left
panel). Using the vIgDTM platform, we have created ICOSL vIgDs
with increased affinity for both ICOS and CD28, of interest given the
important and non-redundant role of each molecule in
costimulation. ICOSL vIgDs provide increased costimulatory axis
blockade compared to CTLA-4 based therapeutics and may act at
multiple stages of organ transplant rejection (below) and other
inflammatory disorders.
• Web: www.alpineimmunesciences.com
• Twitter: @AlpineImmuneSci
mAb
vIgD
V-mAb
e.g., mAb-vIgD
vIgD
Fc
Soluble Fc Fusion
e.g., vIgD-Fc
TIP (Transmembrane Immunomodulatory Protein)
vIgD
Fc
Tumor
Binding
Domain
Tumor-
Localizing
Fc
Checkpoint
Inhibitor
1/2
vIgD Multi-
Checkpoint
Antagonist
CheckpointInhibitor 3/4
The vIgD Platform: Multiple Therapeutic Formats
Figure 2. Multiple Therapeutic Formats for Alpine Immune Sciences vIgDs
0 5 1 0 1 5 2 0 2 5 3 0 3 5
0
2 0
4 0
6 0
8 0
1 0 0
S tu d y D a y s
% S
urv
iva
l
S a line
W T IC O S L -F c
1 s t G e n IC O S L v Ig D
B e la ta c e p t
GVHD Disease Activity Index (DAI)
(Mean + SEM)
0 5 1 0 1 5 2 0 2 5 3 0 3 5
0
1
2
3
4
5
6
7
S tu d y D a y s
Me
an
DA
I S
co
re
-1 2 7 1 2 1 4 1 9 2 3 2 8 3 3
-4 0
-3 0
-2 0
-1 0
0
G v H D S tu d y # 1 : % B o d y W e ig h t L o s s
S tu d y D a y s
Me
an
% B
od
y W
eig
ht
Lo
ss
S a lin e
W T IC O S L -F c
1 s t G e n IC O S L v Ig D
B e la ta c e p t
