Figure 6. DO11.10 cells overexpressing LAG-3 and LK35.2 cells overexpressing PD-L1 were incubated with the indicated mAbor mAb2 while LK35.2 cells were incubated with OVA peptide and then combined. Cells were incubated at 37oC for 24 hoursprior to measuring IL-2 levels in the supernatant by ELISA.

Figure 1. Cell binding of FS118 and controls toHEK293 cells transduced with human LAG-3 asmeasured by flow cytometry

Binding to human LAG-3 expressed on HEK 293 cells

Binding to cynomolgus LAG-3 expressed on HEK 293 cells

Figure 3. Binding of FS118 and controls toHEK293 cells transduced with cynomolgusmonkey LAG-3 as measured by flow cytometry

TUMOUR

APC APC

LYMPHNODE

T-cell

T-cell

APC

--

T-cell

APC

XX

--T-cell

APC

0 .0 1 0 .1 1 1 0 1 0 0 1 0 0 0 1 0 0 0 0

0

2 ,0 0 0

4 ,0 0 0

6 ,0 0 0

8 ,0 0 0F S 1 1 8

Ig G C o n tro l

a n t i-h L A G -3 m A b

m A b o r m A b2

c o n c o n c e n tr a t io n (n M )

Flu

ore

sc

en

ce

DO11.10 T-cell - hLAG-3

LK35.2- hPD-L1

DO11.10 T-cell activation assay

MHCII / OVA

TCR

PD-1

PD-L1

LAG-3

FS118, anti-human mAb²

IL-2

Figure 7. Representative human PBMC SEB (Staphylococcal EnterotoxinB) superantigen activation assay using anti-human FS118 from a donor.Human CD4 T-cells and iDCs were isolated from donor PBMCs. CD4swere expanded, iDCs matured and then both were combined with SEBin presence of titrated test articles. IFNγ was measured following 4 daysincubation.

0 .0 0 0 1 0 .0 0 1 0 .0 1 0 .1 1 1 0 1 0 0 1 0 0 0

0

1 0 0 0 0

2 0 0 0 0

3 0 0 0 0

F S 1 1 8

a n ti-h P D -L 1 m A b

a n t i-h L A G -3 / m o c k

m A b2

a n t i-h L A G -3 / m o c k

m A b2

+ a n t i-h P D -L 1

m A b

Ig G C o n tro l

S E B

m A b o r m A b2

c o n c e n tr a t io n (n M )

h I

FN

g (

pg

/m

l)

FS118 Batch

NumberAssay Run

PBMC Donor

EC50 (nM)95%

Confidence Interval

T001 Assay 1 Donor A 0.16 0.08 to 0.28

T001 Assay 1 Donor B 0.07 0.02 to 0.24

T001 Assay 2 Donor A 0.28 0.16 to 0.46

T001 Assay 2 Donor B 0.14 0.04 to 0.46

CMC-K-M010

Assay 3 Donor C 0.07 0.02 to 0.23

CMC-K-M010

Assay 3 Donor D 0.044 0.02 to 0.11

Table 2. Potency of FS118 Batches Across Multiple Human PBMC Donors.

Binding affinities

Table 1. Binding affinity and kinetics of FS118to human PD-L1 and LAG-3 (Fc-fusion proteins)determined by Surface Plasmon Resonance(SPR).

FS118, anti-human mAb2 potently activates T-cells in vitroFS118, anti-human mAb² binds both human and cynomolgusLAG-3 and PD-L1

DO11.10 CD4 T-cell hybridoma cells specific for ovalbumin were mixed with LK35.2 B lymphoma cells, leading to theactivation and IL-2 release by the DO11.10 cells. Transduced LAG-3 in DO11.10 cells binds to naturally occurring MHC II onthe LK35.2 cells, leading to inhibition of IL-2 release. Inclusion of an anti-LAG-3 mAb or mAb2 to the cells releases the LAG-3inhibition and increases IL-2 secretion. Alternatively, transduced PD-L1 in LK35.2 cells binds to naturally expressed PD-1 onDO11.10 cells resulting in inhibition of IL-2 release that can be relieved by addition of anti-PD-L1 mAb or FS118. Investigationof dual inhibition can be accomplished by incubating LAG-3 expressing DO11.10 cells with PD-L1 expressing LK35.2 cells.

Figure 2. Cell binding of FS118 and controls onHEK293 cells transduced with human PD-L1 asmeasured by flow cytometry

Binding to human PD-L1 expressed on HEK 293 cells

Binding to FcRn

Binding to cynomolgus PD-L1 expressed on HEK 293 cells

Figure 4. Binding of FS118 and controls to HEK293cells transduced with cynomolgus monkey PD-L1as measured by flow cytometry

Figure 5. Fitted sensorgrams for affinity determination.Binding affinity of FS118 to immobilized FcRn asmeasured by Surface Plasmon Resonance (SPR) at pH 6.0.

0 .0 1 0 .1 1 1 0 1 0 0 1 0 0 0

0

5 ,0 0 0

1 0 ,0 0 0

1 5 ,0 0 0

2 0 ,0 0 0

2 5 ,0 0 0

a n ti-h P D -L 1 m A b

F S 1 1 8

Ig G C o n tr o l

m A b o r m A b2

c o n c e n t r a t io n (n M )

Flu

ore

sc

en

ce

0 .0 1 0 .1 1 1 0 1 0 0 1 0 0 0

0

2 ,0 0 0

4 ,0 0 0

6 ,0 0 0

8 ,0 0 0

1 0 ,0 0 0

a n ti-h P D -L 1 m A b

F S 1 1 8

Ig G C o n tr o l

m A b o r m A b2

c o n c e n tr a t io n (n M )

Flu

ore

sc

en

ce

-200

0

200

400

600

800

1000

1200

Re

sp. D

iff

(RU

)

0 80 160 240 320 400 480 560 640 720 800

Time (sec)

Dose response assessment of anti-mLAG-3/PD-L1 mAb² in

MC38 model

Immunophenotyping of tumour-infiltrating lymphocytes following repeat dosing of anti-mLAG-3/PD-L1 mAb²

Figure 9. Prolonged LAG-3 suppression on tumour-infiltrating lymphocytes populations followingrepeat dosing of anti-mLAG-3/PD-L1 mAb² in MC38-OVA model as measured by flow cytometry.The cohorts were administrated when tumours reached 50mm3, with anti-mLAG-3/PD-L1 mAb² orisotype control at 10mg/kg for 3 doses every 2 days. The anti-LAG-3 antibody used for thedetection by flow cytometry doesn’t compete with the anti-mLAG-3/PD-L1 mAb² binding.

2 4 h7 2 h

0

2

4

6

8

1 0

% C

D4

+ L

AG

-3+

2 4 h7 2 h

0

2 0

4 0

6 0

8 0

1 0 0

% C

D8

+ L

AG

-3+

2 4 h7 2 h

0

1 0

2 0

3 0

% C

D4

5+

CD

4+

2 4 h7 2 h

0

1 0

2 0

3 0

% C

D4

5+

CD

8+

LAG

-3 E

xpre

ssin

g T-

cells

CD4+ T-cells CD8+ T-cells

mAb² 1 dose mAb² 2 doses mAb² 3 doses anti-hPD-L1 mAb IgG Control

Anti-mouse mAb² inhibits tumour growth in a syngeneic colon carcinoma model

TUMOUR CELL

TUMOUR

T-cell

TCR

PD-1

PD-L1

LAG-3

LAG-3 / PD-L1 mAb²

CANCER CELL ANTIGEN

MHC II

FS118 BLOCKS LAG-3 ON T-CELLSAND PD-L1 ON TUMOUR CELLS

FS118 REVERSES IMMUNE SUPPRESION AT THE TUMOUR SITE

FS118 TARGETS LAG-3 EXPRESSING T-CELLS IN THE LYMPH NODES AND INHIBITS THE PD-1/PD-L1 MEDIATED SUPPRESSIVE SIGNAL

Human PBMC SEB activation assay

T c e ll a c t iv a t io n a s s a y h u m a n L A G -3 + h u m a n P D -L 1

0 .0 0 1 0 .0 1 0 .1 1 1 0 1 0 0

0

2 0

4 0

6 0

8 0

1 0 0

m A b o r m A b2

c o n c e n tr a t io n (n M )

mIL

-2 (

pg

/m

l)

T c e ll a c t iv a t io n a s s a y h u m a n L A G -3

0 .0 0 1 0 .0 1 0 .1 1 1 0 1 0 0

0

5 0 0

1 0 0 0

1 5 0 0

2 0 0 0

m A b o r m A b2

c o n c e n tr a t io n (n M )

mIL

-2 (

pg

/m

l)

T c e ll a c t iv a t io n a s s a y h u m a n P D -L 1

0 .0 0 1 0 .0 1 0 .1 1 1 0 1 0 0

0

1 0 0

2 0 0

3 0 0

m A b o r m A b2

c o n c e n tr a t io n (n M )

mIL

-2 (

pg

/m

l)

0 1 0 2 0 3 0

0

5 0 0

1 0 0 0

1 5 0 0

D a y s fo llo w in g f ir s t d o s e

Me

an

Tu

mo

ur

Vo

lum

e +

/-

SB

M (

mm

3)

Figure 8a and b. MC38 tumour cellswere injected subcutaneously inC57/Bl6 mice until a palpabletumour formed.

The cohorts were administratedeither with: PBS or isotype controlor anti-mLAG-3/PD-L1 mAb² at 1, 3,10 or 20 mg/kg

Red arrows represent three dosesinjected at day 0, day 3, and day 6post-randomisation. X axis showstime (days) following the first dose.Y axis shows tumour volume (mm3).

1000nM FS118

100nM FS118

10nM FS118

Decrease in IL-2 production Increase in IL-2 production

0 .0 0 0 1 0 .0 1 1 1 0 0 1 0 0 0 0

0

1 0 0

2 0 0

3 0 0

4 0 0

5 0 0

m A b o r m A b2 c o n c e n tr a t io n (n M )

Flu

ore

sc

en

ce

F S 1 1 8

an ti-h L A G -3 m A b

Ig G C o n tro l

FS118 anti-hLAG-3/mock mAb² + anti-hPD-L1 mAb anti-hPD-L1 mAb anti-hLAG-3/mock mAb²

anti-hLAG-3 mAb + anti-hPD-L1 mAb anti-hLAG-3 bm1 + anti-hPD-L1 bm1 LALA anti-hLAG-3 mAb IgG Control

h IF

Nγ

(pg

/mL)

Dual blockade of PD-L1 and LAG-3 with FS118, a unique bispecific antibody, induces T-cell activation with the potential to drive potent anti-tumour immune responses.

Matthew Kraman, Natalie Fosh, Katarzyna Kmiecik, Katy Everett, Carlo Zimarino, Mustapha Faroudi, Mateusz Wydro, Alexander Koers, Lesley Young, Michelle Morrow, Jacqueline Doody, Mihriban Tuna & Neil Brewis

F-star, Cambridge, United Kingdom

SITC 2017 | NOVEMBER 8-12 | POSTER P348

LAG-3 PD-L1

KD (nM) 0.06 1.00

Ka (1Ms) x 106 6.30 0.38

Kd (1/s) x 10-4

3.50 3.90

IgG Control

anti-mLAG-3/PD-L1 mAb² at 1mg/kg

anti-mLAG-3/PD-L1 mAb² at 3mg/kg

anti-mLAG-3/PD-L1 mAb² at 10mg/kg

anti-mLAG-3/PD-L1 mAb² at 20mg/kg

Anti-mLAG-3/PD-L1 mAb² profoundly impacts early tumour establishment

0 5 1 0 1 50

2 0 0

4 0 0

6 0 0

8 0 0

1 0 0 0

1 2 0 0

1 4 0 0

P B S

0 5 1 0 1 50

2 0 0

4 0 0

6 0 0

8 0 0

1 0 0 0

1 2 0 0

1 4 0 0

Ig G C o n t r o l

0 5 1 0 1 50

2 0 0

4 0 0

6 0 0

8 0 0

1 0 0 0

1 2 0 0

1 4 0 0

1 m g / k g

0 5 1 0 1 50

2 0 0

4 0 0

6 0 0

8 0 0

1 0 0 0

1 2 0 0

1 4 0 03 m g / k g

0 5 1 0 1 50

2 0 0

4 0 0

6 0 0

8 0 0

1 0 0 0

1 2 0 0

1 4 0 01 0 m g / k g

0 5 1 0 1 50

2 0 0

4 0 0

6 0 0

8 0 0

1 0 0 0

1 2 0 0

1 4 0 02 0 m g / k g

CONCLUSIONS

FS118 binds to both LAG-3 and PD-L1 both on human and cynomolgus transduced HEK293 cells

FS118 potently activates T-cells both in the D011.10 T-cell and human PBMC superantigen activation assays

Anti-mLAG-3/PD-L1 mAb² shows anti-tumour activity at low doses with an indication of dose-response in an MC38 tumour model

LAG-3 suppression is observed on CD4+ and CD8+ T-cells in mouse tumours following single or repeat dosing of anti-mLAG-3/PD-L1 mAb²

BACKGROUNDDespite advances with therapies targeting the PD-1/PD-L1pathway, many patients are refractory or relapsefollowing treatment. LAG-3 expression on exhausted T-cells and T-regulatory cells (Tregs) in the tumour may beresponsible for this resistance and provides a rationale forco-treatment with antibodies targeting LAG-3 and PD-L1.

An alternative approach is the development of a bispecificantibody encompassing binding sites for two antigens.FS118 is a bispecific antibody targeting LAG-3 and PD-L1that provides dual pathway blockade with the potential todrive unique biology via co-binding of PD-L1 and LAG-3. mAb² - 150kDa

LAG-3 BINDING SITE

PD-L1 BINDING SITE

MATERIALS AND METHODSAn anti-hLAG-3/PD-L1 mAb² bispecific antibody, termed FS118, was engineered by introducing a distincthuman LAG-3 binding site into the constant region of an anti-hPD-L1 IgG1 molecule.

FS118 was evaluated in vitro for antigen binding and de-repression of LAG-3and PD-L1 function in both a D011.10 T-cell activation system and a super-antigen stimulated peripheral blood mononuclear cells (PBMC) assay.

As FS118 is not functional in mouse assay systems, a surrogate wasgenerated for use in mouse tumour models. Anti-tumour activity of themurine-specific molecule was assessed in vivo in an MC38 mouse tumourmodel and associated immunophenotypic changes were evaluated using flowcytometry.

Tota

l T-c

ells