Novel biparatopic TIM3 antibody effectively blocks multiple inherent ligands and activates anti-tumor immunity
Yuji Mishima*, Kanto Nakajima, Mamoru Shiraishi, Haruka Matsumura, Takahiko Aramaki, Noriko Matsumoto, and Norihiro Nakamura*Email contact:

BrightPath Biotherapeutics Co., Ltd., Kawasaki Research Laboratory, Kawasaki, Japan

Gal-9PS

1.16E-04

2.78E-10(A) Epitope classification of TIM3 antibodies. The antibody groups
connected by arrows do not compete with each other in binding to TIM3.
(B) The biparatopic antibody has increased binding affinity to recombinant
TIM3 molecule with a significant reduction in the dissociation rate constants
as compared to the original homodimeric antibodies.

Biparatopic antibody strongly blocks theinteraction of TIM3 to both galectin-9 and PS

15

100Gal-9

CEACAM1Gal-9 (+)(A) The biparatopic antibody inhibited the binding between human TIM3 andhuman Gal-9 more potently than the pre-developed TIM3 antibodies.
(B) The biparatopic antibody completely blocked binding between PS and TIM3
at the similar concentration as the previously developed antibodies.
(C) The biparatopic TIM3 antibody completely inhibited exogenous Gal-9induced apoptosis to primary CD4+T cells. Data are shown as mean of two
independent donors.

IFNγ+ / CD8+ T cells

Human TIM-3
immunoglobulin V domain

Expected binding form of biparatopic antibody; The epitope of scFv ofTIM3 antibody clone A (shown in blue) was confirmed by analyzing crystal
structure of scFv-TIM-3 complex. The structure of scFv of clone B (shownin yellow) was predicted from the primary amino acid sequence in silico.

Fold Change (vs Isotype)210

BpAb A-BGal-9 binding site

PS binding site

nsCD8+ T cells*Novartis

Anti-TIM3 antibodies were generated by immunizing micewith a purified recombinant human TIM3 protein and TIM3expressing mammalian cell line. Phage display libraries
were constructed using cDNAs of splenocytes and lymph
node cells of the immunized mice, then subjected to thebiopanning using recombinant TIM3 proteins.
After analyzing specificities and affinities to the TIM3 protein,scFvs obtained were classified by epitope bin and inhibitory
effects on TIM3 binding to the multiple ligands. The scFvs
were converted to scFv-Fc to generate biparatopicantibodies (BpAbs).

anti- TIM3 scFv
(clone B)

anti- TIM3 scFv
(clone A)

3IsotypeT cells

Methods

IFNγ (pg/mL)

Cytokine production of antigen stimulated PBMCs: PBMCs were stimulatedwith CMV antigen peptides in the presence of test antibodies. The amount ofTNFα and IFNγ produced in the culture supernatant was quantified. Only thebiparatopic antibody significantly enhanced cytokine production of both TNFαand IFNγ. (* p<0.05, ** P<0.01)

Biparatopic TIM3 antibody increases
Interferon-γ producing CD8-positive T cells

Gal-900

*

2.0**

*

1.5

1.00.50.0

BpAb A-B

4.18E+0550

Clone B

BpAb A-B(clone A & B)

100 1000

200

Clone A

KD (M)

10100

400

Novartiskd (1/s)

1150

BpAb A-B

ka (1/Ms)*

Ab (nM)Ab (nM)

Gal-9 (-)

Biparatopic
Ab (scFv-Fc)

0.01 0.1

100 1000(C) Gal-9 induced
apoptosis inhibition**

Clone B

3.20E-07

10600

Clone A

2.43E-02

BpAb A-B

01200

Novartis

7.58E+04

Agenus00.01 0.1

INFγ production

IsotypeClone B

Symphogen

TNFα (pg/mlL)

1.47E-0850

BpAb A-B

6.82E-03

Novartis

Clone B

4.63E+05

BpAb A-B

TNFα production

AnaptyClone A

Clone A

Novartis

No Ab

Gal-9

Group 3KD (M)AnaptyFold Change (vs Isotype)

CEACAM1

Group 4kd (1/s)

Isotype50

Lily100

BpAb A-B

apoptoticcells

Group 1ka (1/Ms)

100

Novartis

Tumor cells

Clone B

Clone A

Homodimeric
Ab (scFv-Fc)

Isotype

Isotype

Dendriticcells

Group 5

Group 2(B) Affinity of biparatopic antibody
to recombinant TIM3

(B) PS binding inhibition

No AbHMGB1(A) Epitope binning of TIM3
antibodies(A) Gal-9 binding inhibition

Isotype

Configuration of biparatopic TIM3 antibody

Binding (%)

T cell immunoglobulin and mucin domain-containing protein 3(TIM3) is a part of modules expressed on dysfunctional or
exhausted T cells as well as dendritic cells. TIM3 has beenreported to have multiple ligands including galectin-9 (Gal-9),
phosphatidylserine (PS), CEACAM-1 and HMGB1, which bindto different regions on the extracellular domain of TIM3. Mostof the TIM3 antibodies developed to date are intended toinhibit PS that binds to the pocket in TIM3 immunoglobulin Vdomain. Gal-9 binds to carbohydrate motifs on the opposite
side of PS-binding site in immunoglobulin V domain andthereby induces cell death in TIM3+ T cells. We report herein
novel antibodies that block TIM3 binding to multiple ligands
including these two important ligands simultaneously.

Biparatopic TIM3 antibody promotes cytokine
production from antigen-stimulated T cells

Biparatopic antibody strongly blocks
Galectin-9 binding to TIM3 molecules

PS positive cells (%)

Results

Binding (%)

Backgroundmishima_y@brightpathbio.com

Isotype#235

SEB stimulation assay : Effect of the TIM3 biparotopic antibody on antigen
stimulation by SEB was evaluated. In the presence of the biparatopic TIM3
antibody, the total CD8+ T cells number was significantly increased
compared to isotype control (1.80-fold increased). In addition, IFNγ-positiveCD8+ T cell number was further increased up to 2.32-fold compared toisotype control. (* p<0.05, ** P<0.01)

Conclusion• This study demonstrates the successful development ofa novel biparatopic antibody against human TIM3.
• The biparatopic antibody has a higher binding affinity tohuman TIM3 and completely blocked both Gal-9 and PS
ligand binding to TIM3 molecules.
• The biparatopic antibody increased the number of IFNγ
producing CD8+T cells by antigen stimulation andpromoted cytokine production of TNFα and IFNγ.
• The biparatopic antibody shows the advantages overconventional TIM3 antibodies and potentiallymanipulated for the development of human monoclonal
antibodies for therapeutic treatment of cancer.

Disclosure

Mishima Y.: Employee of BrightPath Biotherapeutics
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