RESEARCH POSTER PRESENTATION DESIGN © 2015

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Introduction

Results

Arcus Biosciences, Inc., 3928 Point Eden Way, Hayward, CA 94545, USA

Authors: Ashok D, Piovesan D, Zhao X, Singh H, Walters MJ, Young S, Seitz L

Development of a Robust, Simplified Method to Measure Receptor Occupancy in Peripheral Blood from Patients Treated With a Novel Anti-PD-1 Agent, AB122

Exhausted T-cells express high levels of immune

checkpoint proteins, including programmed cell death-1

(PD-1) receptor. Preclinical and clinical data support the

role of PD-1 and its ligand, programmed cell death

ligand 1 (PD-L1), in promoting tumor evasion by

curtailing immune responses. In a Phase 1 clinical trial of

the anti-PD-1 monoclonal antibody AB122, we

determined receptor occupancy (RO) in peripheral blood

T-cells using a directly conjugated competitive antibody

method and the previously established method using

biotinylated anti-human IgG4.

Figure 1. Negative regulation of cytotoxic T-cells mediated by binding

of the receptor PD-1 to its ligand PD-L1 on multiple cell types.

MethodsRO was evaluated using 2 methods: (1) saturation binding

(using a biotinylated anti-hIgG4 for the detection of

AB122, by the previously published method1) and (2)

direct competition (via commercially available anti PD-1

antibody) that is competitive with AB122. Both methods of

RO were optimized to identify proliferating cells by

determining intra-cellular Ki-67+ cells within lymphocyte

sub-populations.

Figure 2. A multi-color flow cytometry panel was developed to

identify PD-1 expression on total CD3+ lymphocytes as well as

CD3+CD8+ (cytotoxic T-cells) and CD3+CD8- (surrogate markers for

CD4+ helper T-cells) using both methods of RO determination on

PBMCs and WB.

Figure 5: AB122 spike-in into healthy volunteer whole blood, donors

A (left) and B (right) and their respective expression of PD-1 on CD8+

cells using both methods of RO determination displays complete RO

with ~10 nM of AB122. Open symbols (isotype).

Robust Multi-Color Flow Cytometry Assay for Determination of PD-1 RO in PBMCs and WB

Comparable PD-1 Expression in Fresh and Freeze/Thaw PBMCs

Figure 4: Directly conjugated anti-PD-1 antibody (blue – isotype,

red – staining) was used to determine RO in whole blood in the

presence of a dose-titration of AB122 (A). A four-parameter global

fit curve (B) of a dose-response to AB122 by 2.5, 1.25 and 0.625 µg

of anti-PD-1 antibody provides an IC50 for AB122 of ~1 nM in-vitro.

Figure 3: AB122 spike-in dose-dependent signal is observed in

freshly isolated (closed symbols) and freeze/thaw PBMCs (open

symbols) of healthy volunteers and shows comparable PD-1

detection. Data is representative of three independent runs.

10 nM AB122 Completely Blocks PD-1 in Human Whole Blood In-vitro

AB122 Exhibits Complete Target Coverage in Dose-Escalation Cohorts (80/240 mg, Q2W)

Both Methods of RO Determination are Compatible with Ki-67 Staining

Figure 6: RO determined using the AB122 saturation binding and

competitive antibody method in AB122 dosed subjects in the dose-

escalation phase. Subjects A-C (80 mg Q2W), subjects D-I (240 mg

Q2W). Please refer to poster # P673 at this meeting for additional

clinical trial information.

Figure 7: Representative plot of CD8+Ki-67+ PBMCs (blue-isotype,

red-staining) from AB122 dosed patient at baseline (left) and post-

dose (right) by both assay protocols of RO determination display

comparable levels of Ki-67+ cells.

PBMCs

Whole

Blood

(WB)

AB122 Dose-Dependent Competition is Observed in Whole Blood

• AB122 exhibits excellent PD-1 target inhibition.

• Both methods of RO determination produce consistent

results and are compatible with intra-cellular Ki-67

staining.

Conclusions

A)

B)

References1Brahmer JR, Drake CG, Wollner I, et al. Phase I study of

single-agent anti-programmed death-1 (MDX-1106) in

refractory solid tumors: safety, clinical activity,

pharmacodynamics, and immunologic correlates. J Clin

Oncol. 2010;28(19):3167-75.

0 16 116 0 16 1160

20

40

60

80

% P

D-1

+o

f C

D8

+

CompetitiveAntibody

SaturationBinding

AB122 [nM]

0 0.7 5 16 0 0.7 5 160

5

10

15

20

25

AB122 [nM] Spike-in

%P

D-1

+ o

f C

D3

+

137 nM AB122

137 nM hIgG4 Iso

Competitive Antibody Saturation Binding

% A

B122

+ o

f C

D3

+

0.1 1 10 1000

20

40

60

80

100

% R

O

PD

-1+ (

of

CD

3+)

0.25 g IC50 : 0.9 nM0.125 g IC50 : 0.7 nM0.06 g IC50 : 0.7 nM

Concentration ofAnti-PD-1 antibody

AB122 [nM]

Dose-Response of AB122 and RO Determination Using

a Directly Conjugated Anti-PD-1 Antibody

0 1 2 15 29 43 55 57 850

50

100

150

200

% R

O

PD

-1+

(o

f C

D3

+)

RO Using AB122 Saturation Binding

0 1.4 13 137 0 1.4 13 1370

10

20

30

40

% P

D-1

+o

f C

D8

+

CompetitiveAntibody

SaturationBinding

AB122 [nM]137 nM AB122

137 nM hIgG4 Iso

0 1 2 15 29 43 55 57 850

40

80

120

% R

O

PD

-1+

(o

f C

D3

+)

A CB D E F G H I

Time Post-Dose (Days)

RO Using A Competitive Antibody