n TUMOUR APC APC LYMPH NODE 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 Brewis F-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-L1 pathway, many patients are refractory to or relapse following treatment. LAG-3 expression on exhausted T cells and T- regulatory cells (Tregs) in the tumour may be responsible for this resistance and provides a rationale for co-treatment with antibodies targeting LAG-3 and PD-L1. An alternative approach is the development of a bispecific antibody encompassing binding sites for two antigens. FS118* is a bispecific antibody targeting LAG-3 and PD-L1 that provides dual pathway blockade with the potential to drive 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 by introducing a distinct human LAG-3 binding activity into the constant region of an anti-hPD-L1 IgG1 molecule. FS118 was evaluated in vitro for antigen binding and de-repression of LAG-3 and PD-L1 function in a D011.10 T cell activation system. FS118 was also assessed in a PBMC (Peripheral Blood Mononuclear Cells) SEB (Staphylococcal Enterotoxin B) assay and in a human CD8 specific MHC I restricted antigen recall assay. Anti-tumour activity of a murine-specific molecule, anti-mLAG-3/PD-L1 mAb 2 , was evaluated in vivo in the MC38 mouse tumour model and associated immunophenotypic changes were evaluated using flow cytometry. 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 the same 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 way repeated 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 is representative 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 and LK35.2 cells overexpressing PD-L1 were incubated with the indicated mAb or mAb 2 while LK35.2 cells were incubated with OVA peptide and then co-cultured. Cells were incubated at 37 o C for 24 hours prior to measuring IL-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 B lymphoma 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 mAb 2 to the cells releases the LAG-3 inhibition and increases IL-2 secretion. Transduced PD-L1 expressed by LK35.2 cells binds to endogenous PD-1 on DO11.10 cells resulting in inhibition 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 with PD-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 and LAG-3 (Fc-fusion proteins) determined by Surface Plasmon Resonance (SPR). LAG-3 PD-L1 K D (nM) 0.06 1.00 Ka (1Ms) x 10 6 6.30 0.38 Kd (1/s) x 10 -4 3.50 3.90 Characterisation of FS118 Binding to FcRn LAG-3 expressing DO11.10 plus PD-L1 expressing LK35.2 0.001 0.01 0.1 1 10 100 0 20 40 60 80 100 mAb or mAb 2 concentration (nM) mIL-2 concentration (pg/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 50mm 3 mice were administrated with one, two or three 200μg intraperitoneal injections of anti-mLAG-3/PD-L1 mAb 2 or controls for 3 doses every 2 days. At designated timepoints following dosing mice were sacrificed and tumours and spleens processed for flow cytometry. It should be noted that the anti-LAG-3 antibody used for the detection by flow cytometry doesn’t compete with the anti-mLAG-3/PD-L1 mAb² binding. mAb 2 = 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 expressing T cells CD4+ T cells CD8+ T cells Total T cells Anti - mLAG - 3/PD - L1 mAb² Inhibits Tumour Growth and Mediates Pharmacodynamic Effects in a Syngeneic Colon Carcinoma Model 24h 48h 72h 0 5 10 15 Time following last dose (hours) CD8 : Treg Ratio **** ** *** *** ** * * Figure 7. The ratio of LAG-3 negative CD8:T regulatory (Tregs) lymphocytes was assessed by flow cytometry following administration of anti-mLAG-3/PD-L1 mAb 2 in MC38 OVA syngeneic tumours (as described in figure 6). mAb 2 = 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 10 20 30 0 500 1000 1500 Time following first dose (days) Tumour volume (mean +/- SEM, mm 3 ) Figure 5a and b. Female C57/Bl6 mice were subcutaneously injected with MC38 tumour cells until a palpable tumour formed and randomised to treatment cohorts. Eight days after tumour cell implantation, the mice were administrated intraperitoneally with either: PBS, isotype control (10mg/kg) or anti-mLAG-3/PD-L1 mAb² at 1, 3, 10 or 20 mg/kg. Red arrows represent three doses injected at day 0, day 3, and day 6 post-randomisation. X axis shows time (days) following the first dose. Y axis shows tumour volume (mm 3 ). Anti-mLAG-3/PD-L1 mAb² profoundly impacts early tumour establishment 0 5 10 15 0 200 400 600 800 1000 1200 1400 PBS 0 5 10 15 0 200 400 600 800 1000 1200 1400 IgG Control 0 5 10 15 0 200 400 600 800 1000 1200 1400 1mg/kg 0 5 10 15 0 200 400 600 800 1000 1200 1400 3mg/kg 0 5 10 15 0 200 400 600 800 1000 1200 1400 10mg/kg 0 5 10 15 0 200 400 600 800 1000 1200 1400 20mg/kg 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 stimulated with a pool of MHC Class I-restricted peptides (cytomegalovirus, Epstein Barr virus, influenza virus and tetanus toxin) and cytokines (interleukin [IL]-7 and IL-15) in the presence of FS118 or control articles. After 7 days samples were washed free of peptides and rested for 4 days. Cultures were then harvested and re-stimulated with peptides for 24 hours following which surface levels of CD107a and intracellular IFNγ were assessed on the CD8+ T cells by flow cytometry. A total of 6 donors were assessed as summarised in table 2. Data for anti-hLAG-3/mock mAb² and anti-hPD-L1 mAb treatments not shown. 0.78 1.56 3.13 6.25 12.50 25.00 50.00 100.00 0.78 1.56 3.13 6.25 12.50 25.00 50.00 100.00 0 1 2 3 4 Concentration (nM) CD107a+ IFN + (Fold change to hIgG Control) 0.78 1.56 3.13 6.25 12.50 25.00 50.00 100.00 0.78 1.56 3.13 6.25 12.50 25.00 50.00 100.00 0 1 2 3 4 Concentration (nM) CD107a+ IFN + (Fold change to hIgG Control) 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 >1.5 fold change for 2 or more different concentration points. Combination is defined as the co-administration of anti-hLAG-3/mock mAb² and anti-hPD-L1 mAb. 24h 48h 72h 0 10 20 30 % CD45+ CD8+ Time following last dose (hours) 24h 48h 72h 0 2 4 6 8 10 % CD4+ LAG-3+ Time following last dose (hours) * * * ** * ** 24h 48h 72h 0 20 40 60 80 100 Time following last dose (hours) % CD8+ LAG-3+ *** ** *** *** ** *** *** * *** 24h 48h 72h 0 5 10 15 20 % CD45+ CD4+ Time following last dose (hours) ** mAb² 1 dose mAb² 2 doses mAb² 3 doses anti-PD-L1 mAb IgG Control 0 100 200 300 400 0 100 200 300 Time (Seconds) Response Units (RU) hFcRN 3.4 nM hFcRN 6.9 nM hFcRN 13.8 nM hFcRN 27.7 nM hFcRN 55.5 nM hFcRN 111 nM hFcRN 222 nM hFcRN 445 nM hFcRN 890 nM hFcRN 1780 nM hFcRN 0 nM pH 7.4 Figure 1. Fitted sensorgrams for affinity determination. Binding affinity of FS118 to immobilized FcRn as measured by Surface Plasmon Resonance (SPR) at pH 6.0. Red arrow shows pH being restored to pH 7.4. Tumour Volume (mm 3 ) Tumour Volume (mm 3 ) Tumour Volume (mm 3 ) 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 0.001 0.01 0.1 1 10 100 0 10 20 30 40 50 Concentration (nM) IFN concentration (ng/ml) **** **** **** **** **** 0.001 0.01 0.1 1 10 100 0 10 20 30 40 50 60 Concentration (nM) IFN concentration (ng/ml) FS118 anti-hLAG-3/mock mAb² anti-hPD-L1 mAb IgG Control anti-hLAG-3/mock mAb² + anti-hPD-L1 mAb
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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
2 0
3 0
4 0
5 0
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
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