Discovery of a PD-1 Checkpoint Agonist Antibody for ... · Agonist antibodies that negatively...
Transcript of Discovery of a PD-1 Checkpoint Agonist Antibody for ... · Agonist antibodies that negatively...
Discovery of a PD-1 Checkpoint Agonist Antibody for
Autoimmune/Inflammatory Disease
Marilyn Kehry, Ph.D.
Vice President, Cell and Functional Biology
March 2nd 2020
PD-1 is an Inhibitory Checkpoint Molecule that Down-modulates
T Cell Responses
• Immune checkpoint receptor-ligand interactions are essential for
down-regulating immune responses and maintaining self-tolerance
• Functional antagonist antibodies to PD-1 and CTLA-4, major
checkpoints on activated T cells, enhance existing immune
responses and are approved therapeutics for immuno-oncology
• Genetic mutations in the PD-1 pathway have been shown to
increase susceptibility to various autoimmune and inflammatory
diseases*
• We hypothesize that many human autoimmune diseases occur due
to dysregulated PD-1 signaling, leading to uncontrolled T cell
responses
• Agonist antibodies to PD-1 that mimic the function of natural
ligands and augment PD-1 signaling have the potential to suppress
human autoimmune/inflammatory diseases and reinstate tolerance
PD-1 agonist discovery for autoimmune/inflammatory disease 2
APCPVR PVRL2
OX40L
CD27L
CD137L
B7RP1
CD80/CD86
CD80/CD86
PD-L1/PD-L2
PD-L1
HVEM
ALARMINS
PVR PVRL2
MHC II
Co
-Sti
mu
lato
ryC
o-I
nh
ibit
ory
MHC
CD226
OX40
CD27
CD137
ICOS
CD28
CTLA-4
PD-1
CD80
BTLA
TIM-3
TIGIT
LAG-3
TCR
T cell
*Okazaki T and Honjo T (2007) Int. Immunol. 19:813.
Agonist antibodies that negatively signal are expected to restore natural immune modulation
PD-1 Agonist Antibody Discovery ANB030 is a Humanized IgG1,κ Hybridoma-derived PD-1 Agonist Antibody
3
Immunization
Fusion
sPD-1-Fc
PD-1+ cells
PD-L1 non-
blocking
hybridomas
Purified antibodies:
cell binding, PD-1 ligand
blocking, agonist activity
PD-1+ cell binders
screened for PD-1
ligand competition
+
ANB030
Lead candidate
CDR-grafted Ab
Hybridoma
ANB030 is PD-L1 non-blocking, optimized for affinity and functional activity, and has no ADCC activity
Optimization for Tm,
affinity, developability
Humanization
PD-1 agonist discovery for autoimmune/inflammatory disease
Optimization of ANB030 VH Sequence for Stability and Affinity
PD-1 agonist discovery for autoimmune/inflammatory disease 4
Optimized for thermal stability and affinity with 2 VH substitutions
Additionally de-risked in 28-day accelerated stability and pre-formulation studies at 100 mg/ml
Humanized
VH
Humanized
VL
CDRH1 CDRH2 CDRH3
CDRL1 CDRL2 CDRL3
Saturating Mutagenesis at 4 VH Positions
Affinity by KinExA
ANB030 Variant
Antibody
KD
Human
PD-1
KD
Cynomolgus
PD-1
Fab Tm
Mouse chimeric 4.3 nM n.d. n.d.
CDR-grafted 0.19 nM 1.00 nM 61.2°C
CDR-grafted
Optimized
(ANB030)
0.075 nM 0.45 nM 66.1°C
ANB030 Binding to PD-1 Does Not Compete with PD-L1 Binding
5
Ligand non-blocking is desirable to avoid potential antagonist activity
PD-1 agonist discovery for autoimmune/inflammatory disease
Model of the Epitope Bound by PD-1 Agonist Antibody ANB030
PD-1 agonist discovery for autoimmune/inflammatory disease 6
• Regions on PD-1 bound by
ANB030 were identified by
hydrogen-deuterium exchange
(pink, blue) and PD-1 surface
point mutations (red)
• Both datasets generated a
consistent model of the ANB030
epitope
ANB030 binds PD-1 on a more membrane-proximal region opposite the PD-L1 binding site
PD-1
sans loop
PD-1 mutations that
eliminate ANB030
binding
ANB030 in Solution Potently Inhibits Whole Blood Tetanus
Toxoid Recall Response
PD-1 agonist discovery for autoimmune/inflammatory disease 7
0 . 0 1 0 . 1 1 1 0 1 0 0 1 0 0 0
0
5 0 0
1 0 0 0
1 5 0 0
C o n t r o l s
A n t i b o d y ( n M )
IF
N
(
pg
/m
l)
IgG4 Isotype
nivolumab
No Ab
Antibody (nM)
1500
1000
500
0
IFNγ
(pg
/ml)
0.01 0.1 1 10 100 1000
PD-L1-IgG1 Fc
Tetramer 0 . 0 1 0 . 1 1 1 0 1 0 0 1 0 0 0
0
5 0 0
1 0 0 0
1 5 0 0
A n t i b o d y ( n M )
IF
N
(
pg
/m
l)
ANB030
IgG1 Isotype
PD-1 Agonist ANB030
No Ab
Antibody (nM)
0.01 0.1 1 10 100 1000
1500
1000
500
0
IFNγ
(pg
/ml)
4 d
Whole
blood
Tetanus Toxoid
Anti-PD-1
IFNγ
PHA-
activatedCD4+ T
cells
IL-2
Anti-CD3ε & Ab
coat
PD-1 agonist antibody inhibits tetanus toxoid IFNγ recall response in whole blood
PD-1 antagonist antibody nivolumab shows no inhibition
ANB030 Functional Agonist Activity in Whole Blood is
Dependent on FcγR Engagement
PD-1 agonist discovery for autoimmune/inflammatory disease 8
ANB030 human IgG2, IgG4, or IgG1(L234A,L235A) isotypes lack agonist activity in solution
0 . 0 0 1 0 . 0 1 0 . 1 1 1 0
0
5 0 0
1 0 0 0
1 5 0 0
W T I g G 1 A N B 0 3 0
A n t i b o d y ( n M )
IF
N
(
pg
/m
l)
0 . 0 0 1 0 . 0 1 0 . 1 1 1 0
0
5 0 0
1 0 0 0
1 5 0 0
I g G 2 I s o t y p e A N B 0 3 0
A n t i b o d y ( n M )
IF
N
(
pg
/m
l) IgG2 Isotype
No Ab
ANB030
IgG1 Isotype
ANB030 IgG2
No Ab
1500
1000
500
0
IFNγ
(pg
/ml)
0.001 0.01 0.1 1 10
Antibody (nM) Antibody (nM)
1500
1000
500
0
IFNγ
(pg
/ml)
0.001 0.01 0.1 1 10
Wild Type IgG1
ANB030
IgG2 Isotype
ANB030
4 d
Whole
blood
Tetanus Toxoid
Anti-PD-1
IFNγ
PHA-
activatedCD4+ T
cells
IL-2
Anti-CD3ε & Ab
coat
PD-1 agonist discovery for autoimmune/inflammatory disease 9
Alopecia areata is an immune-mediated form of hair loss resulting from breakdown of immune privilege
that is driven by keratinocyte and melanocyte antigen-specific T cells producing IFNγ
• NKG2D+CD8+ infiltrate into hair follicle root
sheaths
• Excessive IFNɣ production by activated T cells
leads to loss of hair follicle immune privilege
• Abnormal expression of MHC class I and II
molecules
• Subsequent destruction of hair follicle cells &
hair loss
Healthy Hair Follicle
• Low MHC class I
• Locally immune
suppressed
Alopecia areata Hair Follicle
Alopecia Areata Is An Immune-Mediated Disease
Inhibition of IFNγ Production by ANB030 in Human Alopecia
Areata PBMCs Stimulated with Keratinocyte Peptide Antigens
PD-1 agonist discovery for autoimmune/inflammatory disease 10
1 10 100
0
50
100
150
200
Concentration (nM)
IFN [
pg
/ml]
(% o
f sti
mu
late
d s
am
ple
wit
h
no
test
art
icle
)
IgG1 Isotype
ANB030
0
******* ****
++++
*** P < 0.0005**** P < 0.0001++++ P < 0.0001
1 10 100
0
50
100
150
200
Concentration (nM)
IFN [
pg
/ml]
(% o
f sti
mu
late
d s
am
ple
wit
h
no
test
art
icle
)
IgG1 Isotype tet
0
PD-L1 tet
****
**** ****
++++
**** P < 0.0001++++ P < 0.0001
ANB030PD-L1-IgG1 Fc
Tetramer
IgG1 Isotype
TetramerIgG1 Isotype
200
150
100
50
0
0 1 10 100
Antibody (nM)
0 1 10 100
Antibody (nM)
IFNγ
(% o
f n
o A
NB
03
0) 200
150
100
50
0IFNγ
(% o
f n
o A
NB
03
0)
Keratinocyte Antigen Pool 1
Secreted IFNγ (Day 5)
Normalized Results from 12 Donors
5 d
PBMCs
Anti-PD-1
Keratinocyte Ags
IFNγ
Alopecia areata is an immune-mediated form of hair loss resulting from breakdown of immune privilege
that is driven by keratinocyte and melanocyte antigen-specific T cells producing IFNγ
PD-1 Signaling in T Cells
PD-1 agonist discovery for autoimmune/inflammatory disease 11Bardhan K, Anagnostou T and Boussiotis VA (2016) Front. Immunol. 7:550.
ANB030 Induces SHP2 but not SHP1 Recruitment to PD-1 after
Activation of Jurkat PD-1 Cells
PD-1 agonist discovery for autoimmune/inflammatory disease 12
49PD-1
62SHP2
kDa
SHP162
PD-1 IP
+αCD3 +αCD3
2 minutes 10 minutes
WB
SHP2
αCD3
αPD-1
beads
+
PD-1 Jurkat
Lyse cells,
add anti-PD-1 beads to
isotype samples for
PD-1 immunoprecipitation
SHP2
ANB030 had no effect on signaling pathways in the absence of T cell activation
NSG/Hu-PBMC Graft vs. Host Disease Model
13
1. Isotype control IgG1
2. Anti-PD-1 agonist IgG1 (ANB030)
3. CTLA-4-Ig (positive control)
Day 0: Irradiation
0.9 x 107 hu PBMC i.v.
mAb dosing 24-hr-post PBMC
Weight and GvHD assessment 3X weekly
d.7 d.14 d.21 d.28 d.35 d.42
NSG mice
n=12/group
FACS FACS
Endpoints:
• Weight loss (-20%)
• Death
• GvHD scores
Groups:
Dosing days
PD-1 agonist discovery for autoimmune/inflammatory disease
FACS
PD-1 Agonist Antibody ANB030 is Efficacious in an Acute
Xenogeneic Graft vs. Host Disease Model
14
Treatment Median
Survival
Isotype 20 days
ANB030 39.5 days
Efficacy of ANB030 in the model is dependent on its IgG1 isotype
PD-1 agonist discovery for autoimmune/inflammatory disease
0 1 0 2 0 3 0 4 0
0
5 0
1 0 0
G v H D K a p l a n - M e i e r P l o t
S t u d y D a y
Pe
rc
en
t
su
rv
iv
al
I g G 1 I s o t y p e
A N B 0 3 0
L a s t d o s i n g d a y
* p = 0 . 0 2*
Summary:
PD-1 Agonist Discovery for Autoimmune/Inflammatory Disease
• A functional agonist anti-PD-1 antibody that down-regulates antigen-specific immune
responses and lacks antagonist activity has been discovered and optimized
• ANB030 is a humanized IgG1/κ anti-PD-1 agonist antibody that is non-blocking for PD-
L1 binding and requires Fcγ receptor engagement for its functional activity in solution
• Signaling mechanism studies show similar PD-1-dependent effects for ANB030 and
PD-L1-Fc
• ANB030 demonstrated efficacy in a xenogeneic NSG-Human-PBMC graft vs. host
disease model
• An IND for ANB030 has been filed and Phase 1 clinical trial initiation is anticipated in
H1 2020
• Anti-PD-1 antibodies that mimic activity of natural ligands and down-modulate T cell
responses have the potential to restore and maintain immune balance in autoimmune
and inflammatory diseases
15PD-1 agonist discovery for autoimmune/inflammatory disease
Acknowledgments
• Cell Biology: Allison Rooks, Jack Manorek, Natasha Del Cid, Eric Hare, Nina Schommer
• Protein Sciences: Larry Altobell, Robert Morse, Greg Gold
• Molecular Biology & Protein Expression: Geoff Tomlinson, Brandon Dolan
• Stephen Parmley, V.P. Molecular Biology & Protein Sciences
• Martin Dahl, Executive Director Cell & Translational Biology
• Margaret Marino, V.P. Project Management
16PD-1 agonist discovery for autoimmune/inflammatory disease