Preclinical Characterization of AB154, a Humanized α-TIGIT Antibody, For

Use in Combination Therapies

Anderson AE1, Becker A1, Yin F1, Singh H1, Zhao X1, Seitz L1, Udyavar A1, Stanton R2, Walker NPC1, Tan JBL1

1 Arcus Biosciences, Inc., Hayward, CA2 Stanton Biosciences, Newbury Park, CA

IntroductionTIGIT (T-cell immunoreceptor with Ig and ITIM domains) is an

inhibitory receptor expressed on NK and CD8+ T cells, as well as

immunosuppressive Tregs. TIGIT and DNAM-1 (CD226) are paired

receptors which compete for shared ligands CD155 (PVR) and

CD112 (Nectin-2). As malignancies progress, high TIGIT expression

often occurs alongside the up-regulation of other immune checkpoint

proteins and markers of T cell exhaustion such as PD-1. Shifting

CD155/CD112 from an unfavorable binding with immunosuppressive

TIGIT towards the more productive DNAM-1 interaction promotes T

cell and NK cell activation. We have developed AB154 to inhibit

and modulate the tumor microenvironment towards a more effective

anti-cancer response.

Figure 2. TIGIT and PD-1 expression were derived from RNAseq in

The Cancer Genome Atlas (TCGA) database. Numbers indicate log2

transformed expression of counts per million. Tumors with high

expression of both PD-1 and TIGIT (red), high TIGIT expression but

low PD-1 (blue) and lower levels of both TIGIT and PD-1 (black) are

shown. (B.) TIGIT and PD-1 expression on select tumor types

compared to solid normal tissue. *p ≤ 0.05. **p ≤ 0.01. ***p ≤ 0.001.

****p ≤ 0.0001.

PD-1 Expression Strongly Correlates

with TIGIT in Tumor Infiltrating T cells

Similar AB154 Potency on Peripheral

Blood Lymphocytes Isolated from

Healthy Donors and NSCLC Patients

AB154 Binding to Human TIGIT Blocks

Interaction with CD155

Conclusions

Figure 3. Tumor infiltrating lymphocytes (TILs) analyzed from

dissociated tumor samples revealed a strong correlation between

PD-1 and TIGIT expression on the surface of intratumoral T cells. (A.)

TIGIT is highly expressed on CD8+ and CD4+ CD25+ tumor infiltrating

T cells in different cancers. Right panel shows representative TIGIT

expression levels. (B.) Immunophenotyping of tumor infiltrating T cells

highlights significant TIGIT and PD-1 double positive cells.

Representative data from NSCLC shown.

B.

A.

Figure 4. (A.) In a CHO cell line over-expressing human TIGIT,

AB154 binds with sub-nanomolar affinity (0.35 nM). (B.) Binding of

soluble CD155-Fc to TIGIT was abrogated in the presence of AB154

with an IC50 of 0.69 nM.

Inhibition of TIGIT and PD-1 / PD-L1

Increases IFNγ Production in DC-MLR

AB• 154 is a humanized monoclonal antibody that potently

inhibits binding of human TIGIT to CD155.

TIGIT• and PD-1 expression are correlated in many tumor

types and are often co-expressed on tumor infiltrating

lymphocytes (TILs).

In• combination, AB154 enhances T cell activation relative to

α-PD-1 (AB122) or α-PD-L1 alone in MLR settings.

AB• 154 has sub-nanomolar potency on peripheral blood

lymphocytes derived from both healthy donors and NSCLC

patients.

Flow• cytometry will be used to monitor receptor occupancy

on CD8+ and CD4+ T cells, regulatory T cells, NK cells, and

NKT cells in Phase 1 dose escalation studies for AB154

monotherapy and AB154 / AB122 combination therapy.

Figure 6. Whole blood was obtained from healthy donors (n=3) and

non-small cell lung carcinoma (NSCLC) patients and assessed by

flow cytometry. (A.) Patients and healthy donors had similar

abundance of total lymphocytes, including CD8+ and CD8- T cells, NK

cells and NKT cells. TIGIT expression was also consistent in these

populations, as measured using saturating levels of a commercially

available α-TIGIT antibody. (B.) Fluorophore-conjugated AB154 was

used to directly determine binding affinity in whole blood, with

equipotency observed on lymphocytes isolated from healthy donors

and cancer patients.

Figure 5. Combinatorial settings were assessed using mixed

lymphocyte reactions (MLRs) consisting of CD4+ T cells and

monocytic DC cultures. Inhibition of TIGIT in combination with an (A.)

α-PD-L1 or (B.) α-PD-1 antibody (AB122) resulted in increased IFN-g

production relative to monotherapy. *p ≤ 0.05. **p ≤ 0.01.

Figure 7. (A.) TIGIT receptor occupancy was determined using

saturating levels (>EC90) of a commercially-available human α-TIGIT

antibody that binds competitively with AB154. Therefore, the apparent

IC50, as measured in this assay, is higher than the binding affinity of

AB154 shown in Figure 6B. (B.) In human whole blood, ex vivo

addition of AB154 achieved complete inhibition of TIGIT. Similar

receptor occupancy was observed between cancer patient and

healthy donor blood samples at all AB154 spike-in concentrations.

A Robust Assay to Measure Target

Engagement by AB154

TCGA Analysis of TIGIT and PD-1

Expression on Human Tumors

Figure 1. TIGIT binds to CD155 and results in decreased activation

of the TIGIT-expressing immune cells. AB154 blockade of TIGIT

allows CD155 to bind DNAM-1, favoring T cell and NK cell activation.

0

2 0

4 0

6 0

8 0

C D 4+

C D 2 5+

Pe

rc

en

t

T IG IT+

P D -1-

T IG IT+

P D -1+

T IG IT-

P D -1+

T IG IT-

P D -1-

0

1 0

2 0

3 0

4 0

5 0

C D 4+

C D 2 5-

Pe

rc

en

t

T IG IT+

P D -1-

T IG IT+

P D -1+

T IG IT-

P D -1+

T IG IT-

P D -1-

0

2 0

4 0

6 0

8 0

C D 8+

T C e lls

Pe

rc

en

t

T IG IT+

P D -1-

T IG IT+

P D -1+

T IG IT-

P D -1+

T IG IT-

P D -1-

Expression Level

+/-

++++

Cancer Cell type Cell Surface Markers TIGIT

TCRb+ CD4+ CD25- +/-

TCRb+ CD4+ CD25+ ++++

TCRb+ CD8+ ++++

CD11b+ CD14+ HLA-DR+ -

CD11b- CD14- HLA-DR+ -

TCRb+ CD4+ CD25- +/-

TCRb+ CD4+ CD25+ ++++

TCRb+ CD8+ ++++

CD11b+ CD14+ HLA-DR+ -

CD11b- CD14- HLA-DR+-

TCRb+ CD4+ CD25- +/-

TCRb+ CD4+ CD25+ ++++

TCRb+ CD8+++++

CD11b+ CD14+ HLA-DR+ -

CD11b- CD14- HLA-DR+-

TCRb+ CD4+ CD25- +/-

TCRb+ CD4+ CD25+ ++++

TCRb+ CD8+++++

CD11b+ CD14+ HLA-DR+ -

CD11b- CD14- HLA-DR+ -

SKCM

CRC

NSCLC

KIRC

T cells

Antigen Presenting Cells

T cells

Antigen Presenting Cells

T cells

Antigen Presenting Cells

T cells

Antigen Presenting Cells

0

1 0 0 0

2 0 0 0

3 0 0 0

IFN

-g (

pg

/ml)

A B 1 5 4 -P D -L 1 A B 1 5 4 +

-P D -L 1

Ig G 1

* *

A124

0

1 0 0 0

2 0 0 0

3 0 0 0

IFN

-g (

pg

/mL

)

*

Ig G 4 Ig G 1 Ig G 4 +

Ig G 1

A B 1 2 2 A B 1 2 2 +

A B 1 5 4

T cell and NK cell

Activation

CD155

TIGIT

DNAM

T cell and NK cell

Activation

CD155

TIGIT

DNAM

AB154

-2 0

0

2 0

4 0

6 0

8 0

1 0 0

L y m p h o c y t e P o p u la t io n s

Pe

rc

en

t

H e a lth y

N S C L C

C D 8+

T c e lls(T c e lls % )

C D 8-

T c e lls(T c e lls % )

N K T( ly m p h o c y t e s % )

N K( ly m p h o c y t e s % )

T o ta l

L y m p h o c y te s( s in g le t s % )

T c e lls( ly m p h o c y t e s % )

0

2 0

4 0

6 0

8 0

1 0 0

T IG IT + (% )

TIG

IT+

%

H e a lth y

N S C L C

C D 8+

T c e lls

C D 8-

T c e lls

N K T N KT o ta l

L y m p h o c y te s

T c e lls

A.

B.

B.

A.

Healthy Donors (n=3) NSCLC Patients (n=3)

0 .0 1 0 .1 1 1 0 1 0 0

0

1 0

2 0

3 0

4 0

A B 1 5 4 -A F 6 4 7 (n M )T

IGIT

+ %

(To

tal

Ly

mp

ho

cy

tes

)

M e a n E C 50 = 0 .1 3 n M

0 .0 1 0 .1 1 1 0 1 0 0

0

1 0

2 0

3 0

4 0

A B 1 5 4 -A F 6 4 7 (n M )

TIG

IT+

%

(To

tal

Ly

mp

ho

cy

tes

)

M e a n E C 50 = 0 .1 4 n M

0 .1 n M 1 n M 1 0 n M 1 0 0 n M

0

5 0

1 0 0

L y m p h o c y t e R e c e p t o r O c c u p a n c y

A B 1 5 4 S p ik e -In C o n c e n tra tio n s

Re

ce

pto

r

Oc

cu

pa

nc

y (

%)

N S C L C

H e a lth y

A. B.

A.

B.

A. B.

0 .0 1 0 .1 1 1 0 1 0 0

0

1 0 0 0 0 0

2 0 0 0 0 0

3 0 0 0 0 0

4 0 0 0 0 0

A B 1 5 4 (n M )

MF

I

h T IG IT

E C 50 = 0 .3 5 n M

-2 0 2 4

-2

-1

0

1

2

M e d ia n T IG IT C P M (L O G 2 )

Me

dia

n P

D-1

CP

M (

LO

G2

)

B L C A

B R C A

C E S C

C O A D

E R / P R + B R C A

E S C A

G B M

H E R 2 + B R C A

H N S C

K I R C

K I R PL I H C

N S C L C (a d e n o )

L U S C

O V

P A A D

P R A D

R E A D

S A R C

S K C M

S TA D TN B CU C E C

U C S

R2 = 0 .6 5 , p < 0 .0 0 0 1

0 .0 1 0 .1 1 1 0 1 0 0

0

2 0 0

4 0 0

6 0 0

8 0 0

1 0 0 0

A B 1 5 4 (n M )

MF

I

IC 50 = 0 .6 9 n M

AB154

Human

TIGIT

-1 0

-5

0

5

1 0

G B M

CP

M

* n s

P D -1 T IG IT-1 0

-5

0

5

1 0

K IR P

CP

M

* * *

P D -1 T IG IT-1 0

-5

0

5

1 0

B R C A

CP

M

P D -1 T IG IT

* * * * * * *

-1 0

-5

0

5

1 0

C O A D

CP

M

n s * *

P D -1 T IG IT

-1 0

-5

0

5

1 0

H N S C

CP

M

n s * * * *

P D -1 T IG IT-1 0

-5

0

5

1 0

K IR C

CP

M

* * * * * * * *

P D -1 T IG IT-1 0

-5

0

5

1 0

T N B C

CP

M

* * * * *

P D -1 T IG IT

-1 0

-5

0

5

1 0

N S C L C (a d e n o )

CP

M

* * * * * * * *

P D -1 T IG IT

S o lid N o rm a l T is s u e T u m o r