PowerPoint PresentationFigure 6. DO11.10 cells overexpressing LAG-3 and LK35.2 cells overexpressing...
Transcript of PowerPoint PresentationFigure 6. DO11.10 cells overexpressing LAG-3 and LK35.2 cells overexpressing...
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
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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
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-200
0
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Re
sp. D
iff
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)
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
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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
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8 0 0
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1 4 0 03 m g / k g
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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