1221

BP1202-NF2, a novel ADCC-enhancing CD39 antibody, induces destruction of regulatory T cells
and enhances cytotoxic T lymphocyte induction.
Toshifumi Obonai, Haruka Ban, Noriko Matsumoto, Yuji Mishima, Motoya Mie and Norihiro Nakamura
Kawasaki Research Laboratories, BrightPath Biotherapeutics Co., Ltd., Kawasaki, Japan.
Introduction

Fr. I
CD4+FOXP3CD8+ PD-1+Tim-3+KD (M)Antibody

CD39 expression(binding sites/cell)CD8+

CD39 expression in Tregs and exhausted
T cells of tumorinfiltrating lymphocytes was higher than PBMCs in 3 NSCLC
patients (Stage I or IV).

BP1202-NF2 profiles
BP1202KD (M)KD (M)

RecombinantTreg

7.11 x 10-10

3.98 x 10-9ー

4.06 x 10-10

BP1202-NF2

4.07 x 10-10

BP1202-NF3

4.31 x 10-10

BP1202-NF1

BP1202-NF3

10

Mogamulizumab
Ipilimumab
0.00010.01

1FcγRIIIa

1.82 x 10ー-6BP1202

1.63 x 10

BP1202-NF1
BP1202-NF2

8.28 x 10-7
4.11 x 10-7

Anti-CCR4 antibody
mogamulizumab and antiCTLA4 antibody ipilimumab
are reference drugs for Treg
depletion.
40

 Correlation of ADCC activity andaffinity against FcγRIIIa wasobserved among BP1202(unmodified IgG1) and BP1202NF1~3 (altered glycosylation).
 BP1202-NF2 showed the highest
ADCC activity for Treg and potent
killing of CD39+ cancer cells inADCC assay.

-9EC50BP1202-NF2ng/mL
12.3

Mogamulizumab

79.6

Ipilimumab> 10

EC50 = 9.5 ng/mL

Both BP1202 and BP1202-NF2 show high affinity for recombinanthuman CD39 and Tregs. BP1202 (unmodified IgG1) and BP1202NF1~3 (altered glycosylation) inhibit cellular CD39 activity to thesame extent.

Anti-human CD39 antibodies were screened by sorting B cells of mice
immunized with human CD39 protein. The clones that inhibited theenzyme activity of CD39 were humanized on IgG1. A glycosylation
modification was then introduced to the antibody during its production.
Binding to human CD39 and Treg was evaluated via surface plasmon
resonance (SPR) or flow cytometry. Functional assay was performedby Promega CD16 (V/F variants) ADCC signaling assay. Treg depletionand CTL induction were assayed using peripheral blood mononuclear
cells (PBMCs) from healthy donors. All human samples were handled
under the guidelines of BrightPath Biotherapeutics Co., Ltd.
Institutional Review Board (IRB) with an approved protocol (#ERD-01
and #BP20200312).

Ag specific CTLs induced by BP1202-NF2
inhibit cancer cell growth3020
10non-exhausted
00.000001

0.00010.01

Ab (ug/mL)

1100

PBMC effectors were used in ADCC
reporter assays on Tregs or cytotoxic
ADCC assays on CD39-positive OAW-42(Human Ovary Adenocarcinoma) .

+Method

Healthy donor PBMCs cultured with test antibodies (10 µg/mL) at 37℃ with 5% CO2 for 5 days.
Mean±SE（N=3）. ****p<0.0001, ***p<0.001 and **p<0.01 vs IgG1 control (Tukey’s)

BP1202-NF2 selectively depletes CD39hi T cells

Release of adenosine-induced
immunosuppression via CD39 inhibitionµg/mL
no antibody
BP1202-NF2－0.1
110CD39hi
4.35%
0.20%
0.16%
0.09%

CD4 T

 Mogamulizumab (moga) andipilimumab (ipi) partiallydepleted only effector Tregs
but not CD39hi Tregs.

Control IgG1

100

OAW42 (E/T=10)

BP1202-NF3 7.60 x 10-7Mogamulizumab 1.56 x 10-7

Representative data from 3 NSCLC patients (stage I or IV) are shown.

Antibody

BP1202-NF2

20

ADCC (%)

NSCLC TIL

BP1202-NF1

Ab (μg/mL)

CD8+PD-1-Tim-3-

NSCLC PBMC

3000.000001

CD8+ PD-1+

HD PBMCBP1202CD39int
9.62%
4.34%
3.16%
2.89%

CD39hi
7.78%
0.87%
0.62%
0.34%

CD8+ T# of CMV specific CD8+ T cells(cells/well)Fr. II40

PD-1+Tim-3+CD39250000

PD-1+Tim-3+CD39+

20000015000010000050000
0noAbIgG1 NF2 moga ipi

Growth inhibiton of tumor cells
intCD39
13.3%
10.0%
9.01%
8.68%

Healthy donor PBMCs cultured with test antibodies at 37℃ with 5% CO2. After 6 days culture,
frequencies of CD39hi and CD39int population among CD4+ and CD8+ T cells were analyzed byflow cytometry.

BP1202-NF2 selectively depleted CD39hi population of CD4+ T cells andCD8+ T cells in ex vivo cultured human PBMCs.

nsA375-CMV-RFP (cells/well)

ADCC (fold change)

Treg00
10 000015 000
020 00000

 BP1202-NF2, a novel glycosylation-modified monoclonal antibody(mAb) targeting human CD39, has both antibody-dependent cellularcytotoxicity (ADCC) and inhibiting activity against CD39 enzyme.

Fr. III0

 Selective elimination of tumor-infiltrating Tregs is expected to reinvigorate anti-tumor immunity.

 BP1202-NF2 depleted CD4+
CD39hi FoxP3+ Tregs andeffector Tregs (CD4+CD45RA-FoxP3hi)
significantly. These effects
were restored by anti-CD16(FcγRIII） antibody.

Activated Treg5050

 CD39 (ENTPD1), which contributes to immune regulation throughadenosine-signaling in the tumor microenvironment (TME), isexpressed on Tregs and plays a role in Treg function.

BP1202-NF2 depletes CD39hi Tregs

ADCC activity

CD39 Expression in Tumor-Infiltrating
Lymphocytes

 Regulatory T cells (Tregs), suppressive immune cells, are known tosuppress anti-tumor immune response.

Contact: takeshige_k@brightpathbio.com✱✱✱✱120,000

BP1202-NF2 enhanced induction of non-exhausted CTLs.

✱✱✱✱80,000

After Treg depletion with antibodies (10 µg/mL), PBMCs (HLAA*02:01) were cultured with CMV-expressed malignant melanomacell line A375 for 5 days. Mean±SE（N=3）. ****p<0.0001 vs IgG1control (Tukey’s).

40,0000After Treg depletion with antibodies (upper), PBMCs (HLA-A*02:01)
were cultured with CMV-peptide and second antibodies (lower) for
CTL induction for 11 days.

IgG1 NF2 moga ipi

BP1202-NF2 treated PBMCs inhibited in vitro cancer
growth significantly.

Conclusion

ATP-+++IgG1 IgG1 NF2 BP1202 (10 µg/mL)(IgG4)

ATP-+++IgG1 IgG1 NF2 BP1202 (10 µg/mL)(IgG4)

BP1202-NF2 released the immunosuppression on CD8+ T cells
caused by ATP.

The humanized anti-CD39 antibody with glycosylation modification, BP1202-NF2 potently inhibits CD39 enzyme and enhances its
antibody-dependent cellular cytotoxicity (ADCC) activity. BP1202-NF2 almost completely depletes CD39hi Tregs with a high ADCC
activity, which subsequently enhances CTLs induction and significantly inhibits cancer cell growth. Our results suggest that BP1202-NF2
modulates the TME to promote immune response in human tumors via Treg depletion and inhibition of CD39 enzymatic activity.