A novel therapeutic antibody against CD73 that ameliorates the tumor microenvironment andimproves the efficacy of cancer immunotherapy.

987PT. Obonai, H. Ban, Y. Mishima, N. Matsumoto, M. Tsukahara, R. Kuniyoshi, M. Mie, N. Nakamura
Kawasaki Research Laboratories, BrightPath Biotherapeutics Co., Ltd., Kawasaki, Japan.

(B)

RU

Blue ; Nucleus, Red ; BP1200_002
Cell ; MDA-MB-231

Figure 2. Binding of BP1200_002 to human and cynomolgus CD73 (A) and the kinetics
against recombinant human CD73 (B).

806040030120

Incutabion (min)240IL-2 (pg/mL)

3000
2000

Incubation (min)240

w/o mAb or ATP01020304010

1

10.

0.
0101

HCC827

1.0

Figure 4. Internalization ofBP1200_002 into tumor cells,
MDA-MB-231 (A). BP1200_002
downregulates CD73 on cellsurface of MDA-MB-231 (B) andhuman CD4+/CD8+ T cells (C).

0.0

BP1200_002

3MEDI9447

1050

100Time (h)150050300

10203040Days after innoculation

BP1200_002+ αPD-L1

αPD-L1

BP1200_002

BP1200_002+ αPD-1

Isotype Control

w/oPBMC

αPD-1Isotype Control

BP1200_002 is a humanized anti-CD73 antibody that
attenuates the AMP hydrolysis activity of CD73 as a noncompetitive inhibitor without hook effect.

•BP1200_002 is internalized into tumor and T cells and results inthe downregulation of CD73 expression on the cell surface.

•BP1200_002 enhances the proliferation, cytotoxicity, andcytokine production of T cells under the TME condition.

•The combination of BP1200_002 and immunocheckpoint
antibody for cancer treatment will be a promising regimen inclinical practice.

Isotype Control

2020200•

41.510400

ConclusionmAb (µg/mL)

mAb (µg/mL)

2.050200600

Figure 7. Effects of BP1200_002 in combination with PD-1 blockers on tumor growth in mice.
(A) NOD mice were transplanted with human PBMC and NCI-H292 cancer cell line, thentreated by BP1200_002 (0.2 mg/body, BIW) in combination with anti-PD-L1 antibody (0.2mg/body, BIW). (B) B6 mice were transplanted with MC-38 expressing hCD73, then treatedby BP1200_002 (10 mg/kg, BIW) in combination with anti-PD-1 (3 mg/kg, QW) . Data arepresented as means ±SEM.

Figure 5. Human CD4+ and CD8+ T cells were stimulated with anti-CD3/CD28 beads in thepresence of ATP. BP1200_002 enhanced both proliferation of T cells (A) and production ofcytokines (B).

MEDI9447

Tumor volume (mm3)Tumor volume (mm3)Isotype Control

1
0.
000

101

1
0.

0.
01

0.
001

00.5

120

MEDI9447

3000

0400

800

Days after innoculation

4000

1000

600

010

BP1200_002

5000

1000

2.5

MEDI9447

30

1

2000

mAb (µg/mL)

BP1200_002

BP1200_002

00.1

IL-2

7000

0.
0Cells/Well

00.01

6000

NCI-H292

204005000406020100000.001

BP1200 augments the cytotoxicity of T cells.

6080

20080

Incubation (hr)100

MEDI9447% CD73 expression

KD (M)
2.04x10-10% CD73 expression

Time (sec)kd (1/s)
1.14x10-4

1000

CD8+ T cells

BP1200_002

100ka (1/Ms)
5.61x105800

Tumor cells

1000CD4+ T cells

TFNα

Fold change

Recombinant human CD73

(C)

BP1200_002
Isotype control

400600Substrate (μM)

BP1200 internalizes into cells and depletes CD73
on cell surface.

25 µm

00.0001

10mAb (µg/mL)

1Dendritic cells:
Tumor antigenpresentation ↓BP1200 binds human and cynomolgus CD73 withhigh affinity.

CD73 binding

200Figure 3. Inhibition of cellular CD73 (A) and soluble CD73 without hook effect (B).
BP1200_002 is a non-competitive inhibitor of CD73 (C). MEDI9447 is in-house produced
analogues. Adenosine 5’-(α,β-methylene) diphosphate (APCP) is a CD73 small molecule
inhibitor.

Result(B)

000

-20

1000

20000

40000.
0800(A)HEK293T_cyCD73

15000

TNFα (pg/mL)

400600Substrate (μM)

w/o mAb or ATP

5000

102001

6000

20000.1

IFNγ1200.0120000

400Figure 1. Ado is generated by CD39 and CD73 suppresses immune cells, such as T cells, NKcells, macrophages and dendritic cells in TME.

(A) HEK293T_huCD73% of Enzyme activity

40(B)

60

Isotype Control

30000mAb (µg/mL)

Isotype Control

800.001

1000

MEDI9447

10000

00.0001

APCP

100

Isotype Control

60-2020000

1NK cells:
Tumor cell lysis ↓100

Competitive inhibition

120BP1200_002

80

10mAb (nM)

Non-competitive inhibition

120

1400000.

Tumor Microenvironment

0.1

w/o mAb or ATP(B) hCD73-expressing MC38 syngenic model

800

BP1200_002

40000

Isotype Control

0.
01(C)

MEDI9447

6000001

DC00.01% CD73 expression

Tumorcells

100-50100

Macrophages:
M2 polarization ↑5050000

BP1200_002

80000

1TAMATP

11000.
0NK

0.01

100000(A) PBMC-tumor co-transplantation modelCD8+ T cells

CD4+ T cells

Isotype ControlmAb (μg/mL)

T cells:
CD4+ T cell proliferation ↓T cell Treg polarization ↑ATPATP0

MEDI9447

150(A)

00

AMP

50Soluble CD73

0.
0Adenosine

Isotype Control

BP1200_002

IFNγ (pg/mL)

CD73

MEDI9447100(B)Cellular CD73

ΔAMP (μM)

CD4+
T cell CD39

BP1200_002

ΔAMP (μM)

CD8+
T cellCD8+
T cell(A)% Inhibition

CD73 is expressed on certain types of leukocytes and tumor
cells and is known as a poor prognostic factor in severalcancers. CD73 catalyzes conversion of extracellular adenosine
monophosphate (AMP) to adenosine (Ado) which accumulates
in the tumor microenvironment (TME). Ado suppresses
immune activation through Ado receptors on several immune
cells, allowing tumors to escape from the immune surveillance.
Therefore, inhibition of CD73 activity provokes T cell activation
and reverses immune suppression exerted by Ado. Thus, ournovel potent anti-CD73 antibody (BP1200_002) is expected todo provide cancer immunotherapy options.

BP1200 inhibits tumor growth in combination
with immune checkpoint blockades.

BP1200 enhances T cell proliferation andcytokine production.

Cells/Well

BP1200 inhibits the activities of CD73 on cell
membrane and soluble CD73 without hook effect.

Fold change

Introduction

100150Time (h)Figure 6. Augmentation of PBMCs-mediated cytotoxicity against tumor cells by BP1200_002in the presence of ATP. Data are presented as means ±SEM. Both NCI-H292 and HCC827
are human lung cancer cell line.

Disclosure

T. Obonai: Employee of BrightPath Biotherapeutics
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