Becker A, Narasappa, N, Yin F, Zhang K, Park T, …...Becker A, Narasappa, N, Yin F, Zhang K, Park...

RESEARCH POSTER PRESENTATION DESIGN © 2015

www.PosterPresentations.com

CD73i effects in a mono-therapeutic setting were assessed by

CD3/CD28/CD2 T cell stimulation and cytolytic assays.

Combinatorial settings were assessed using mixed lymphocyte

reactions (MLRs). In vivo effects of CD73i + ICI were determined

using syngeneic tumor models. Multiple structurally-related CD73

inhibitors (CD73i) used in this presentation: AB680, A000830,

A001421

INTRODUCTIONDIFFERENTIAL EXPRESSION OF CD73 AND PD-1 INTUMORS

CD73 catalyzes the extracellular generation of adenosine (ADO) from

adenosine monophosphate (AMP). ADO suppresses immune

responses, including those of T cells, NK cells and dendritic cells

through activation of A2aR and A2bR receptors. Exhausted T cells and

NK cells express high levels of several immune checkpoint proteins,

including PD-1 and TIGIT. We present here preclinical data on the

ability of CD73i to reverse effector cell suppression from exposure to

ADO even in the presence of ICI.

METHODS

AB680 Limits the Inhibitory Effect of AMP onCD4+ T Cell Activation

RESULTS AND CONCLUSIONS

CD73 is expressed across a wide range of tumor types, including

those with limited response to anti-PD-1 therapy. CD73i completely

rescued AMP-mediated inhibition of T cell proliferation and effector

function as well as NK cell cytolytic function. AMP abrogated the

enhanced allogeneic CD4+ T cell activation and IFN-γ production

mediated by blocking PD-1/PD-L1 and TIGIT, an effect that was

reversed by CD73i. Mechanistically, addition of AMP in MLRs

repressed expression of activation markers and immune checkpoint

proteins. Thus, activation of the adenosinergic pathway may limit the

efficacy of ICI. TCGA data from anti-PD-1-treated melanoma patients

identified CD73 expression as a negative prognostic factor. Finally, co-

administration of a CD73i with an anti-PD-1 mAb resulted in significant

reduction of tumor volume associated with increases in immune cell

infiltration.

CD73 inhibition, alone or in combination with anti-PD-1 and anti-TIGIT

antibodies, translates into potent enhancement of immune cell

activation in a variety of studies. These data provide a rationale for

clinical evaluation of CD73i + ICI combinations.

AB680 Reverses the Activity of AMP on a-PD-1 (AB122) and a-TIGIT (AB154) Antibodies in MLR

Figure 2: RNASeq data generated from Hugo W et al, (Cell Vol.

165 (1): 35-44, 2016 were used to plot CD73 and A2AR

expression in melanoma patients treated with nivolumab or

pembrolizumab. a-PD-1 responders (CR or PR, n = 13 patients)

and non-responders (PD, n = 13) were shown.

CD73 Inhibitors Enhance the Activity of a-PD-1in B16.F10 Melanoma Model

Figure 6: In-house generated CD73 inhibitor, A000830 or AB680,

together with a-PD-1 antibody (RMP1-14) were dosed when

tumors reached a volume of 50 mm3. Change in tumor volume

(A) associated with changes in the immune cell infiltrates (B) are

shown. Similar to A000830, AB680 also increased efficacy of a-

PD-1 antibody as measured by tumor volume (C) and survival

curves (D). Mice bearing tumors > 1000 mm3 were sacrificed as

per protocol. Pink circles indicates days when a-PD-1 antibody

dosing occurred.

Figure 1: CD73 expression were derived from RNASeq in the

TCGA database and depicted as box and whisker plots (above).

Expression of CD73 compared to PD-1 is shown (bottom)

Opposing Effects of AMP and a-PD-(L)1 on

Immune Modulatory Protein Expression inMLR

Becker A, Narasappa, N, Yin F, Zhang K, Park T, Kalisiak J, Lawson K, Jeffrey J, Powers JP, Schindler U, Walters, MJ, Tan JBL

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

CD73 Inhibitors (CD73i) Reverse the AMP/Adenosine-Mediated Impairment of Immune Effector Cell Activation by Immune Checkpoint Inhibitors (ICI)

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CTLA-4 LAG-3 PD-1 TIM-3 TIGIT 4-1BB ICOS CD28

AMP

a-PDL1

Figure 5: (A) Expression of indicated proteins in the presence of

AMP (top row) or a-PD-L1 antibody (bottom row) compared to

samples treated with isotype were determined. The reversibility

of AMP-mediated immune checkpoint protein expression was

shown using CD73 inhibitors (B). Gray bars represent AMP

alone; green bars represent a-PD-L1 blocking antibody alone,

red bars represent AMP + a-PD-L1 blocking antibody, and blue

bars represent AMP + a-PD-L1 blocking antibody + CD73i. (C)

Global transcriptional changes in MLR with AMP or with AMP +

AB680. Numbers indicate fold change compared to AB122

alone.

Figure 4: AB122 (A) or AB122 and AB154 (B) were

simultaneously added to CD4+ T cells and monocytic DC

cultures containing 100 mM AMP. Addition of AB680

completely reversed the inhibitory effects of AMP.

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AMP AMP + AB680CD39 6.29 1.76

CD73 0.51 1.18

ADA 0.22 1.22

CD26 1.15 1.03

IL2 0.15 0.85

IFNG 0.52 0.69

TNF 1.13 0.69

IL10 2.91 0.96

TGFB 3.66 0.74

TBX21 0.39 0.91

GATA3 1.35 1.13

FOXP3 1.53 1.26

CTLA-4 0.96 1.2

LAG-3 0.72 0.93

PD-1 0.19 0.73

TIM-3 1.63 0.82

TIGIT 1.02 1.08

PDL-1 2 1.08

PDL-2 2.67 1.63

LGALS3 1.93 0.73

IDO-1 1.92 1.14

CSF1 1.19 0.51

Immune

Modulation

AB122

Adenosine

Pathway

Cytokines

Transcription

Factors

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CD73 AND A2AR Expression in a-PD-1 Respondersand Non-responders

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Figure 3: Proliferation (A) and cytokine secretion (B) in the

presence of AMP and varying concentrations of AB680 tested.

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T u m o r T y p e s

RP

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

11. Sarcoma

12. Kidney (papillary)

13. Head and Neck

14. Esophageal

15. Pancreatic

16. Stomach

17. Colorectal

Ovarian1.

Kidney (Clear Cell)2.

Adenocortical3.

Lung (squamous)4.

Bladder5.

Breast6.

Liver7.

Lung 8.(adenocarcinoma)

Prostate9.

Melanoma10.

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CD73 Inhibitors Enhance the Activity ofLymphokine Activated Killer Cells

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Figure 7: Lymphokine activated killer cells (LAKs) were

generated from C57BL/6 splenocytes cultured in IL-2.

Expression of adenosine receptors were assessed by qPCR

(A). LAK cells pre-incubated with A001421 and AMP were co-

cultured with LLC target cells loaded with caspase detection

reagent (B). Cytolytic activity of LAKs were depicted as percent

caspase-positive LLCs.

CTLA-4 LAG-3 PD-1 TIM-3 TIGIT 4-1BB ICOS CD28

CD73 PD-1Sarcoma 1077 19.88

Liver 975.9 16.46

Pancreatic 973.5 32.1

Colorectal 963.5 21.62

Stomach 935.2 51.78

Adeno (lung) 908.9 54.8

Esophageal 821.8 28.28

Head and Neck 664.1 39.91

Clear cell (kidney) 649.1 46.11

Papillary Cell (kidney) 557.1 19.53

Ovarian 342.9 22.94

Breast 299.4 17.94

Bladder 286.9 19.48

Melanoma 280.1 53.49

Squamous (lung) 253.6 43.43

Adenocortical 105.5 3.353