PowerPoint Presentation - f-star.com · Barr virus, influenza virus and tetanus toxin) and cytokines (interleukin [IL]-7 and IL-15) in the presence of FS118 or control articles. After

nTUMOUR

APC APC

LYMPHNODE

T cell

T cell

APC

X

TUMOUR

T cell

TCR

PD-1

PD-L1

LAG-3

LAG-3 / PD-L1 mAb²

CANCER CELL ANTIGEN

MHC II

FS118 BLOCKS LAG-3 AND PD-L1 WITHIN THE TUMOUR ENVIRONMENT

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

Dual blockade of PD-L1 and LAG-3 with FS118, a unique bispecific antibody, induces CD8+ T cell activation and modulates the tumour microenvironment to promote anti-tumour immune responses.

Matthew Kraman, Natalie Fosh, Katarzyna Kmiecik, Katy Everett, Carlo Zimarino, Mustapha Faroudi, Mateusz Wydro, Alexander Koers, Lesley Young, Daniel Gliddon, Michelle Morrow, Jacqueline Doody, Mihriban Tuna & Neil BrewisF-star, Cambridge, United Kingdom

mAb² - 150kDa

LAG-3 BINDING

PD-L1 BINDING

CONCLUSIONS

FS118 binds to LAG-3 and PD-L1 and can activate in a D011.10 T cell assay via co-inhibition of both targets.

FS118 potently activates human CD4+ and CD8+ T cells in a SEB assay and a CD8 T cell antigen-recall assays respectively.

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

Anti-mLAG-3/PD-L1 mAb² treatment reduces LAG-3 expression on CD4+ and CD8+ T cells in mouse tumours.

Anti-mLAG-3/PD-L1 mAb² increases the ratio of LAG-3 negative CD8:Tregs in mouse tumours.

BACKGROUND

Despite advances with therapies targeting the PD-1/PD-L1pathway, many patients are refractory to or relapse followingtreatment. LAG-3 expression on exhausted T cells and T-regulatory cells (Tregs) in the tumour may be responsible forthis resistance and provides a rationale for co-treatment withantibodies 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 by co-binding of PD-L1 and LAG-3.

MATERIALS AND METHODS

An anti-human LAG-3/PD-L1 mAb² bispecific antibody, termed FS118, was engineered byintroducing a distinct human LAG-3 binding activity into the constant region of an anti-hPD-L1 IgG1molecule.FS118 was evaluated in vitro for antigen binding and de-repression ofLAG-3 and PD-L1 function in a D011.10 T cell activation system. FS118was also assessed in a PBMC (Peripheral Blood Mononuclear Cells) SEB(Staphylococcal Enterotoxin B) assay and in a human CD8 specific MHC Irestricted antigen recall assay.

Anti-tumour activity of a murine-specific molecule, anti-mLAG-3/PD-L1mAb2, was evaluated in vivo in the MC38 mouse tumour model andassociated immunophenotypic changes were evaluated using flowcytometry.

FS118 can Potently Activate both primary human CD4 and CD8 T cells in vitro

FS118 enhances an antigen-driven CD8+ T cell response in a subset of donors

T cell

APC

✓

FS118 REVERSES IMMUNE SUPPRESION AT THE TUMOUR SITE

TUMOUR CELL

Figure 3. CD4+ T cells and CD14+ monocyte subpopulations were isolated from PBMCs from 9 healthy donors, and the CD4+ T cells were activated with anti-CD3/CD28 beads, while the monocytes were cultured and differentiated into immature dendritic cells (iDCs). The activated CD4+ T cells and the iDCs from thesame donor were co-cultured for 4 days with SEB in the presence of FS118 or other test antibodies over a range of concentrations from 0.00128 to 100 nM.IFNγ released into the culture supernatant was quantified by ELISA. Significant increases between FS118 versus combination are annotated. Two wayrepeated measures ANOVA with Dunnett’s multiple comparison to FS118 activity are shown * p <0.05, ** p< 0.01, *** p<0.001, ****p<0.0001. Donor 1 isrepresentative of 4 out of 9 donors tested and donor 2 is representative of 5 out of 9 donors tested.

FS118 enhances CD4+ T-cell activation in a human PBMC SEB assay with the potential for greater activity than a combination in a subset of donors

Figure 2. DO11.10 cells overexpressing LAG-3 andLK35.2 cells overexpressing PD-L1 were incubated withthe indicated mAb or mAb2 while LK35.2 cells wereincubated with OVA peptide and then co-cultured. Cellswere incubated at 37oC for 24 hours prior to measuringIL-2 levels in the supernatant by ELISA.

DO11.10 T cell activation assay

DO11.10 CD4 T cells with a TCR specific for ovalbumin were mixed with LK35.2 Blymphoma cells, leading to the activation and IL-2 release by the DO11.10 cells.Transduced LAG-3 expressed by DO11.10 cells binds to MHC II on the LK35.2 cells,leading to inhibition of IL-2 release. Inclusion of an anti-LAG-3 mAb or mAb2 to thecells releases the LAG-3 inhibition and increases IL-2 secretion. Transduced PD-L1expressed by LK35.2 cells binds to endogenous PD-1 on DO11.10 cells resulting ininhibition of IL-2 release that can be relieved by addition of anti-PD-L1 mAb or FS118.Dual inhibition can be assessed by incubating LAG-3 expressing DO11.10 cells withPD-L1 expressing LK35.2 cells with FS118.

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 mAb

IgG Control

Binding affinities

Table 1. Binding affinity and kinetics of FS118 to human PD-L1 andLAG-3 (Fc-fusion proteins) determined by Surface PlasmonResonance (SPR).

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

Characterisation of FS118

Binding to FcRn

L A G -3 e x p r e s s in g D O 1 1 .1 0

p lu s P D -L 1 e x p r e s s in g L K 3 5 .2

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 c

on

ce

ntr

ati

on

(p

g/

ml)

Anti-mLAG-3/PD-L1 mAb² modulates LAG-3 expression on CD4+ and CD8+ tumour-infiltrating lymphocytes (TILs)

Figure 6. LAG-3 expression on tumour-infiltrating lymphocytes populations following repeat dosing of anti-mLAG-3/PD-L1 mAb² in MC38-OVA model as measured by flow cytometry. When subcutaneous tumours reached 50mm3 mice were administrated with one, two or three200μg intraperitoneal injections of anti-mLAG-3/PD-L1 mAb2 or controls for 3 doses every 2 days. At designated timepoints following dosingmice were sacrificed and tumours and spleens processed for flow cytometry. It should be noted that the anti-LAG-3 antibody used for thedetection by flow cytometry doesn’t compete with the anti-mLAG-3/PD-L1 mAb² binding. mAb2 = anti-mLAG-3/PD-L1 mAb². Statistical test:2-way ANOVA * p ≤ 0.05; ** p ≤ 0.01; ***p ≤ 0.001; ****p< 0.0001

LAG

-3 e

xpre

ssin

g T

cells

CD4+ T cells CD8+ T cells

Tota

l T c

ells

Anti-mLAG-3/PD-L1 mAb² Inhibits Tumour Growth and Mediates Pharmacodynamic Effects in a Syngeneic Colon Carcinoma Model

2 4 h 4 8 h 7 2 h

0

5

1 0

1 5

T im e f o llo w in g la s t d o s e ( h o u r s )

CD

8 :

Tre

g R

ati

o

****

**

***

*****

*

*

Figure 7. The ratio of LAG-3 negative CD8:T regulatory (Tregs)lymphocytes was assessed by flow cytometry followingadministration of anti-mLAG-3/PD-L1 mAb2 in MC38 OVAsyngeneic tumours (as described in figure 6). mAb2 = anti-mLAG-3/PD-L1 mAb². Statistical test: 2-way ANOVA * p ≤0.05; ** p ≤ 0.01; ***p ≤ 0.001; ****p< 0.0001

Anti-mLAG-3/PD-L1 mAb² increases the ratio of LAG-3 negative CD8:T regulatory cells within tumours

Anti-tumour activity of anti-mLAG-3/PD-L1 mAb² in the MC38 model

0 1 0 2 0 3 0

0

5 0 0

1 0 0 0

1 5 0 0

T im e fo llo w in g f irs t d o s e (d a y s )

Tu

mo

ur

vo

lum

e (

me

an

+/

- S

EM

, m

m3

)

Figure 5a and b. Female C57/Bl6mice were subcutaneouslyinjected with MC38 tumour cellsuntil a palpable tumour formedand randomised to treatmentcohorts. Eight days after tumourcell implantation, the mice wereadministrated intraperitoneallywith either: PBS, isotype control(10mg/kg) or anti-mLAG-3/PD-L1mAb² at 1, 3, 10 or 20 mg/kg.Red arrows represent threedoses injected at day 0, day 3,and day 6 post-randomisation. Xaxis shows time (days) followingthe first dose. Y axis showstumour volume (mm3).

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

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

Figure 4. Human T cells were isolated from donor PBMCs and stimulatedwith a pool of MHC Class I-restricted peptides (cytomegalovirus, EpsteinBarr virus, influenza virus and tetanus toxin) and cytokines (interleukin [IL]-7and IL-15) in the presence of FS118 or control articles. After 7 days sampleswere washed free of peptides and rested for 4 days. Cultures were thenharvested and re-stimulated with peptides for 24 hours following whichsurface levels of CD107a and intracellular IFNγ were assessed on the CD8+ Tcells by flow cytometry. A total of 6 donors were assessed as summarised intable 2. Data for anti-hLAG-3/mock mAb² and anti-hPD-L1 mAb treatmentsnot shown.

0.7

8

1.5

6

3.1

3

6.2

5

12

.50

25

.00

50

.00

10

0.0

0

0.7

8

1.5

6

3.1

3

6.2

5

12

.50

25

.00

50

.00

10

0.0

0

0

1

2

3

4

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

CD

10

7a

+ I

FN

+

(Fo

ld c

ha

ng

e t

o h

IgG

Co

ntr

ol)

0.7

8

1.5

6

3.1

3

6.2

5

12

.50

25

.00

50

.00

10

0.0

0

0.7

8

1.5

6

3.1

3

6.2

5

12

.50

25

.00

50

.00

10

0.0

0

0

1

2

3

4

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

CD

10

7a

+ I

FN

+

(Fo

ld c

ha

ng

e t

o h

IgG

Co

ntr

ol)

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

FS118

Strictly for personal use -DO NOT POST

AACR 2018 | APRIL 14-18 | POSTER 2719 | DO NOT POST

Donor 1 Donor 2

FS118 > combination > hIgG control

FS118 = combination > hIgG control

FS118 = combination = hIgG control

1/6 2/6 3/6

Donor 1 Donor 2

Table 2. Summary of donor responses (n=6): A response was defined as >1.5fold change for 2 or more different concentration points. Combination isdefined as the co-administration of anti-hLAG-3/mock mAb² and anti-hPD-L1mAb.

2 4 h 4 8 h 7 2 h

0

1 0

2 0

3 0

% C

D4

5+

CD

8+

T im e fo llo w in g la s t d o s e (h o u r s )

2 4 h 4 8 h 7 2 h

0

2

4

6

8

1 0

% C

D4

+ L

AG

-3+

T im e fo llo w in g la s t d o s e (h o u r s )

*

*

*

* *

*

* *

2 4 h 4 8 h 7 2 h

0

2 0

4 0

6 0

8 0

1 0 0

T im e fo llo w in g la s t d o s e (h o u r s )

% C

D8

+ L

AG

-3+

* * *

* *

* * *

* * *

* *

* * *

* * *

*

* * *

2 4 h 4 8 h 7 2 h

0

5

1 0

1 5

2 0

% C

D4

5+

CD

4+

T im e fo llo w in g la s t d o s e (h o u r s )

* *

mAb² 1 dose

mAb² 2 doses

mAb² 3 doses

anti-PD-L1 mAb

IgG Control

0 1 0 0 2 0 0 3 0 0 4 0 0

0

1 0 0

2 0 0

3 0 0

T im e (S e c o n d s )

Re

sp

on

se

Un

its

(R

U)

h F c R N 3 .4 n M

h F c R N 6 .9 n M

h F c R N 1 3 .8 n M

h F c R N 2 7 .7 n M

h F c R N 5 5 .5 n M

h F c R N 1 1 1 n M

h F c R N 2 2 2 n M

h F c R N 4 4 5 n M

h F c R N 8 9 0 n M

h F c R N 1 7 8 0 n M

h F c R N 0 n M

p H 7 .4

Figure 1. Fitted sensorgrams for affinity determination. Bindingaffinity of FS118 to immobilized FcRn as measured by SurfacePlasmon Resonance (SPR) at pH 6.0. Red arrow shows pH beingrestored to pH 7.4.

Tum

ou

rV

olu

me

(m

m3)

Tum

ou

rV

olu

me

(m

m3)

Tum

ou

rV

olu

me

(m

m3)

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

DO11.10 T cell - LAG-3

LK35.2- PD-L1

LAG-3

MHCII / OVA

TCR

PD-L1

IL-2

PD-1

FS118, anti-human mAb²

*FS118 is under option to Merck KGaA

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

0

1 0

2 0

3 0

4 0

5 0

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

IFN

co

nc

en

tra

tio

n (

ng

/m

l)

* * * *

* * * ** * * *

* * * ** * * *

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

0

1 0

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6 0

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

IFN

co

nc

en

tra

tio

n (

ng

/m

l)

F S 1 1 8

a n t i- h L A G - 3 / m o c k m A b ²

a n t i- h P D - L 1 m A b

Ig G C o n t r o l

a n t i- h L A G - 3 / m o c k m A b ² +

a n t i- h P D - L 1 m A b