Bispecific antibodies against TIM-3 and CD39 induce anti-tumor efficacy and immune response by blocking multiple suppressive mechanisms.
Noriko Matsumoto, Yuji Mishima, Kanto Nakajima, Takahiko Aramaki, Noriko Watanabe, Mamoru Shiraishi, Haruka Matsumura, and Norihiro Nakamura
BrightPath Biotherapeutics Co., Ltd., Kawasaki Research Laboratory, Kawasaki, JapanTIM-3

TIM-3• BP1210 completely inhibits the ligand-binding of both galectin-9
and phosphatidylserine. BP1210 enhances the antigendependent activation of T cells, and anti-tumor activity with higher
potency than the reference antibody.
• BP1212 binds and blocks CD39 and TIM-3 simultaneously,
resulting in synergistic effects of TIM-3 and CD39 inhibition on
antigen-stimulated CD8+ T cells.
• BP1210 and BP1212 enhance T cell immunity by ameliorating
the immuno-suppressive tumor microenvironment and provide
the advantages over conventional TIM-3 and CD39 antibodies incancer immune therapy.
Disclosure
Authors are employees of BrightPath Biotherapeutics

2.23E-04

7.34E-10

CloneA (dimer)

2.34E+05

1.44E-03

6.13E-09

CloneB (dimer)

8.87E+04

1.41E-02

1.59E-07TIM-3

Inhibition of the ligand-binding

Fold Change of IFNγ+ CD8 T cells

10010050

IsotypeBP1210
MGB4535000.01 0.1 1 10 100
Ab (nM)

0.01 0.1

1 10 1001000
Ab (nM)(A) Inhibition of Gal-9 binding to TIM-3 by the antibodies
was quantified by ELISA.
(B) Inhibition of PS binding to human recombinant TIM-3
by the antibodies was analyzed by FACS.

ns

isotype ctl

nsBP1210211.0

effector-

1005000.50.0

150050Time (h)1001008060
402000.01 0.10
Isotypectl MGB453
MGB453 BP1210
BP1210Isotype

5,000
4,000
3,000

isotype control
BP12102,000KD (M)KD (M)

BP1212(TIM-3 x CD39)

17.56E-9

5.67E-9

8.31E-9anti-TIM3 (dimer)

N.D.

1.97E-9

2.75E-9anti-CD39 (dimer)

7.04E-9

N.D.

16.84E-9

100,0000110 100 1000
Ab (nM)

Enhanced inhibition of CD39 byBP1212

BP1212 Enhances the expansion ofIFNγ-producing T cells✱✱✱✱✱✱

100on CD39 single+ cells
isotype
BP121250150

100.010(A) Inhibition of PS binding to humanrecombinant TIM-3 was estimated using
ELISA.
(B) Human PBMCs were stimulated with
anti-CD3/CD28 beads in the presence ofATP for 4 days, then analyzed by FACS.

50,000on CD39/TIM-3 double+ cells
isotype
BP121205w/o ATP or Ab
isotype ctl
a-TIM-3
a-CD39
BP1212150,000

1,000

Human CD8⁺ T cells were simulated with CMV-peptide, then co-cultured with colon cancer cell line, SW620
expressing CMV pp65 antigen and RFP. HLA-types of PBMCs and SW620 are A02. (A) The number of RFP⁺ tumorcells were quantified under imaging analyzer. (B) Cytokines were quantified at 48hrs.

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500(B) Cytokine production
200

isotype ctl
anti-TIM-3
BP1212

100

Human PBMCs were stimulated with SEB (Staphenterotoxin B) for 6 days in the presence of antibodies,then the IFNg⁺ CD8⁺ T cells were quantified by FACS.
(** p<0.01, One-way ANOVA and Tukey’s).

250

KD (M)(B) Proliferation of activated-T cells exposed to ATP(A) Inhibition of PS binding

Enhanced cytotoxic activity of CTLs
(A) Growth rate of tumor cells

hCD39+ hTIM-3+
cells

Inhibition of TIM-3 and CD39 functions✱✱3

hTIM-3+cells

activation,
cytotoxicity

BP1210 Enhances IFNγ-producing T cells(A) inhibition of Gal-9 binding (B) Inhibition of PS binding

hCD39+cells

N.D. not detectable% Inhibition

Conclusion

3.03E+05

IL-2 (pg/mL)

ADO: adenosine

BP1210(clone A & B)

hTIM-3 IgV domain

Granzyme B (pg/mL)

Tumor killing ↓KD (M)Number of CD3+ Tcells

activation,
cytotoxicity

IFNγ (pg/mL)

priming ↓Kd (1/s)

Binding (%)

activation,cytotoxicity

TNFα (pg/mL)

T cells

Ka (1/Ms)

BP1210Fold change of tumor cell nubmers

Tumor cells

BP1212

apoptoticcells

Crystal structure ofBP1210/TIM-3 complex

apoptotic cells

0DCs

Monoclonal
TIM-3 AbsPS

Binding (%)

PS

BP1210Binding (%)

Multiple immuno-suppressive mechanisms in tumor have beensuggested to attenuate anti-tumor immunity. Here we developed
bispecific antibodies against TIM-3 and CD39, negative regulators ofanti-tumor immunity, in order to overcome the immune-suppression intumor microenvironment.
BP1210 is a humanized TIM-3 biparatopic antibody (BpAb) thatblocks the ligand binding of TIM-3 including phosphatidylserine andgalectin-9. Monoclonal TIM-3 antibodies in clinical trials inhibit only some,
but not all ligands. Thus, BP1210 potentially improves the efficacy ofTIM-3 blockade.
BP1212 is a humanized bispecific antibody (BsAb) against TIM-3
and CD39. The expression of TIM-3 and CD39 are simultaneouslyinduced in exhausted T cells and DCs and suppress the anti-tumor
activity of T cells and DCs. By inhibiting CD39 in combination with TIM-3
blockade, BP1212 synergically enhances the anti-tumor immunity.
BP1210 and BP1212 that block TIM-3- and adenosinesignaling, provide new and effective therapeutic approaches.

BP1212, a humanized bispecific antibody for TIM-3 and CD39

BP1210, a humanized biparatopic TIM-3 antibody

0.1

1

10100

Ab (nM)

CD39 or CD39/TIM3 co-expressing cells were incubatedwith ATP in the presence of antibodies. The enzyme activityof CD39 was analyzed by quantifying the remaining ATP inthe supernatant.

4IFNγ+ CD8+ T cells(fold change)

BackgroundContact: takeshige_k@brightpathbio.com

✱✱3
210

Isotype aTIM-3 aCD39
BP1212
isotypeaCD39aTIM-3
BP1212aTim-3
controlaCD39
ctrl
aTIM-3
aCD39
combo

combo

PBMCs were stimulated with SEB in the presence ofantibodies for 6 days, then IFNγ+ CD8+ T cells werequantified by FACS. **p<0.01, ****p<0.001