A novel therapeutic antibody against CD73 that ameliorates the tumor microenvironment
and improves the efficacy of cancer immunotherapy

2905

Noriko Matsumoto, Yuji Mishima, Toshifumi Obonai, Haruka Ban , Toshimitsu Itai, Motoya Mie, Norihiro Nakamura.
Kawasaki Research Laboratories, BrightPath Biotherapeutics Co., Ltd., Kawasaki, Japan. *Contact: matsumoto_n@brightpathbio.com

CD4+
T cell

CD39AMP
NKATP

TAM

ATP

Tumorcells

DCTumor Microenvironment

Macrophages:
M2 polarization ↑0.1

110100(B)8060
400Blue ; Nucleus, Red ; BP1200_002
Cell ; MDA-MB-231BP1200_002
MEDI9447
010203040804020030120240

Incubation (min)Incubation (hr)BP1200 binds human and cynomolgus CD73 withhigh affinity.
expressing cells

CD8+ T cells
50000

Cells/Well

RU

40000
30000

100000

MEDI9447

80000

Isotype Control
w/o mAb or ATP

20000

BP1200_002
Isotype control

Time (sec)ka (1/Ms)kd (1/s)
5.61x105 1.14x10-4

KD (M)
2.04x10-10

Figure 2. Binding of BP1200_002 to human and cynomolgus CD73 expressedon tumor or 293 cells(A) and the kinetics to a recombinant human CD73 (B).

00.0001

BP1200_002
MEDI9447
Isotype Control
w/o mAb or ATP

40000

0.001

0.01

0.1mAb (µg/mL)

11000.0001

(B)

0.01

0.1

110mAb (µg/mL)

Figure 5. Human PBMC were stimulated with anti-CD3/CD28 beads in thepresence of ATP. After 96h culture, the number of T cells were determined usingflow cytometry.

100

6×10 54×10 52×10 5Tumor volume (mm3)Fold change of tumor cell number

500

0Pharmacokinetics and Toxicokinetics of BP1200
Table 1. Pharmacokinetics of single intraperitoneal dose of BP1200 in female C57BL/6 mice
DoseCm ax

AUC0→∞

ClVsst1/2

MRT0→∞

mg/kg

μg/mL

hr×mg/mL

mL/hr/kg

mL/kg

hrhr

1091±1524±2

0.41±0.03

119±12201±27290±39

Table 2.Toxicokinetics of single or multiple intravenous dose of BP1200
in female cynomolgus monkeys✱✱ns

3×10 5✱✱ns
ns

2×10 5

1×10 50DayCm ax

AUC0→∞

CLVsst1/2

MRT0→∞

μg・hr/mL
6900
4600

mL/hr/kg
0.7
2.6

mL/kg
52.8
173.3

hr
51.2
42.9

hr
73.9
61.9

0/9 CR0/9 CR

25iv, q1w

1598

222001.1

68.4

41.9

60.4

25iv, q1w

22808

35700

0.7

57.6

57.7

83.2

BP1200_002
BP1200_002 + αPD-1

40CD4+ T cells

Route1228/9 CRCD8+ T cells

40iv, q1w
iv, q1w

αPD-1

(C)

30μg/mL
149
1225/9 CR

30

20

D a y s a fte r in n o c u la tio n

Isotype Control

2010Figure 8. Cured mice treated withcombination therapy of BP1200-002
and anti-PD1 were rechallenged with
the MC-38 expressing hCD73 cells.
Naïve mice were used as a control.
Data are presented as means ±SEM.

mg/kg55400

108/8 CR0Time (h)2000/6 CR

1000

150600ns00.001

50800

8×10 520000

10000

CD73 binding

60000

0Days after innoculation

CD4+ T cells

BP1200_002

150

Cured mice

1500

Dose0BP1200 enhances the proliferation of T cells
in the presence of ATP.

(B) Recombinant human CD73

cyCD73expressing cells

1000

1000

6050501(A)BP1200_002
MEDI9447

100002The combination therapy of BP1200 with ICB
leads tumor regression with increasing thenumber of TILs.

Figure 4. Internalization of CD73 was observed using microscopy (A).
BP1200_002 downregulates CD73 on the cell surface of MDA-MB-231(B) andhuman T cells (C).

Result0.0

BP1200_002
MEDI9447
Isotype ControlFigure 6. Human PBMC were co-cultured with tumor cells in the presence of ATPand antibodies. The tumor cells were quantified using imaging analyzer. BothNCI-H292 and HCC827 are human lung cancer cell lines expressing CD73 onthe cell surface. Data are presented as means ±SEM.

(C) Human T cells

Tumor cells

100

200.5

Naive mice

3Time (h)BP1200 internalizes and depletes
cell surface CD73.
(A)

1.0mAb (nM)

Figure 3. The CD73-expressing MDA-MB-231 cells (A) or recombinant human CD73
protein(B) were incubated with AMP in the presence of antibodies. The enzymeactivity was analyzed by quantifying the remaining AMP in the supernatant.
MEDI9447 is in-house produced analogues.

25 µm(A) hCD73

Fold change of tumor cell number% of Enzyme activity

mAb (μg/mL)

Dendritic cells:
Tumor antigen
presentation ↓Figure 1. Ado generated by CD39 and CD73 suppresses immune cells,such as T cells, NK cells, macrophages and dendritic cells in TME.

100

Number of CD8+ Tcells / gATPNK cells:Tumor cell lysis ↓1

1.5

2000

Cmax, Maximum observed concentration; AUC0→∞, Area under the concentration-time curve from 0 to infinity; CL, Total systemic
clearance; Vss, Volume of distribution at steady state; t1/2, Terminal half life; MRT0→∞, Mean residence time from 0 to infinity

Conclusion• BP1200_002 is a humanized anti-CD73 antibody that attenuates theactivity of CD73 as a non-competitive inhibitor without hook effect.
• BP1200_002 enhances the proliferation, cytotoxicity, and cytokine
production of T cells under the TME condition.

IFNγ+CD8+ T cells

2×10 41×10 40

Number of IFNγ+CD4 Tcells / g

Adenosine

0.01

2.0

The combination therapy of BP1200 with ICB induces
tumor-specific immunity in rechallenge model.

HCC827

Number of IFNγ+CD8 Tcells / g

CD730.000100.01

Cells/Well

CD4+
T cell050

NCI-H292

2.5

Number of CD4+ Tcells / g

T cellT cells:
T cell proliferation ↓Treg polarization ↑50

100

Tumor volume (mm3)CD8+BP1200_002
MEDI9447
Isotype Control150

100% CD73 expression

CD8+T cell(B) Soluble CD73

(A) Cellular CD73

% CD73 expression

CD73, also known as ecto-5’-nucleotidase (NT5E), is a GPIanchored cell surface enzyme expressed on certain types ofimmune cells and tumor cells. This enzyme catalyzes thehydrolytic conversion of extracellular adenosine monophosphate(AMP) to adenosine (Ado) that suppresses tumor-infiltrating
immune cells via the adenosine receptors, allowing tumors toescape the immune system. Here, we introduce a novel antihuman CD73 antibody called BP1200 that strongly blocks thegeneration of Ado by CD73 and thereby reverses immune
suppression in tumors.

BP1200 inhibits the activities of CD73 on the cell BP1200 augments the cytotoxic activity
membrane and soluble CD73 without hook effect. of T cells.

% Inhibition

Introduction

IFNγ+CD4+ T cells

6×10 3

4×10 3

2×10 30Figure 7. C57BL/6 mice were transplanted with MC-38 expressing hCD73, thentreated by BP1200_002 (10 mg/kg, BIW) in combination with anti-PD-1 (3 mg/kg,QW) (A) . The number of TILs were determined on Day 21 after tumor inoculation(B) . IFNγ producibility of T cells were determined by ICS after 4h culture with P/I (C).
Data are presented as means ±SEM. **p<0.005.

• The combination of BP1200_002 and immune checkpoint antibody
significantly suppressed tumor growth and lead long term
immunotherapeutic efficacy.
• BP1200_002 showed good PK/TK profiles without remarkable organ
toxicity in mice and monkeys.
• The combination of BP1200 and immune-checkpoint inhibitors forcancer treatment will be promising therapeutic option in clinicalpractice.
Disclosure
N. Matsumoto: Employee of BrightPath Biotherapeutics

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