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