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0 2 4 6 8 10
0
50
100
150
Antibody concentration (nM)
Norm
aliz
ed fr
eque
ncy
of T
IGIT
+ CD8
+ T
cells
Fortessa HD263
BmaxKd
wuxi109.60.08425
BmaxKd
uprot113.10.09253
BmaxKd
313M32139.51.267
BmaxKd
4.1.D3146.10.8441
BmaxKd
22G2121.90.4507
BmaxKd
31C6114.50.09426
BmaxKd
Arcus104.10.1378
0 5 10 15 20 25
0
500
1000
Days post-tumor cell implant
Tum
or v
olum
e (m
m3 )
hepa 1-6 median TV
PBSaTIGIT mIgG2a
t r e a t m e n t
rando : TV=60 mm
3
p=0,0009
-6 -4 -2 0 2 4
0
1000
2000
3000
4000
5000
Log Ab concentration (nM)
MFI
(CD
155
sign
al)
LGO
iTeos Therapeutics, Gosselies, Belgium Corresponding author: [email protected]
EOS884448 INCREASES IFNγ SECRETION IN T CELLS FROM HEALTHY AND CANCER PATIENTS
EOS884448 BINDS WITH HIGH AFFINITY TO HUMAN AND CYNO TIGIT AND PREVENTS LIGAND BINDING TO TIGIT
SUMMARY
iTEOS SURROGATE Ab SHOWS STRONG ANTITUMOR EFFICACY IN MONOTHERAPY OR COMBO WITH a-PD-1
HIGH TIGIT EXPRESSION ON IMMUNE CELLS, FURTHER INCREASED IN CANCER PATIENTS
ADENOSINE-DRIVEN IMMUNOSUPPRESSIONITEOS SURROGATE Ab INCREASES CD8+ T CELLS AND CYTOTOXICITY SIGNATURES IN VIVO
EOS884448 PREFERENTIALLY DEPLETES TREG IN HUMAN PBMCs FROM HEALTHY DONORS AND CANCER PATIENTS
CONCLUSIONS
TIGIT-DRIVEN IMMUNOSUPPRESSION
NaïveTregMemoryEffector
CD4+ T cells CD8+ T cells
TIGITTCRγ
δ
CD4 naiv
e T ce
lls
CD4 effe
ctor T
cells
CD4 mem
ory T
cells
CD4 Tre
g
CD8 naiv
e T ce
lls
CD8 effe
ctor T
cells
CD8 mem
ory T
cells
0
20
40
60
80
100
% o
f TIG
IT e
xpre
ssio
n%
TIG
IT+
cells
Bind
ing
to C
D8+
T ce
lls
Vehicle aPD-1 aPD-1+aTIGIT mIgG1
aPD-1+aTIGIT mIgG2a
0
10
20
30
CD
8/Tr
eg ra
tio
**p=0.005 **
IC50 = 0.04nM
-3 -2 -1 0 1 2 3
200
250
300
350
400
Log Ab concentration (nM)
IFN
g (p
g/m
l)
EC50 = 0.11nM
a-TIGIT mIgG1
Ctrl a-TIGIT mIgG2a
IFNg+CD8+ T cells
*
% C
D8+ IF
Ng+
in to
tal C
D8+
T ce
lls
Com
petit
ion
with
hCD
155
Func
tiona
l ass
ays
A. Healthy donor CD8+ T cells Cancer patient CD3+ T cells
TIG
IT p
heno
typi
ng
A. Healthy donors B. Cancer patients
ADC
C in
vitr
o as
say
Antitumor efficacy of anti-TIGIT antagonist antibody EOS884448 is mediated by a dual mechanism of action
involving restoration of T cell effector functions and preferential depletion of Tregs. Catherine Hoofd, Julia Cuende, Virginie Rabolli, Julie Preillon, Noémie Wald, Lucile Garnero, Florence Lambolez, Shruthi Prassad, Marjorie Mercier, Florence Nyawame, Margreet
Brouwer, Erica Houthuys, Véronique Bodo, Xavier Leroy, Michel Detheux and Gregory Driessens.
• T cell Immunoreceptor with Ig and ITIM domains (TIGIT) is a negativecostimulatory receptor that inhibits Teff and NK cell function and marks a highlysuppressive Treg subset.
• TIGIT ligands belong to the PVR/nectin family, among which PVR (CD155) showsthe highest affinity and is commonly expressed on antigen presenting cells (APC)and tumor cells.
• CD226, a co-stimulatory receptor also expressed on NK and T cells, competeswith TIGIT for PVR binding but with a lower affinity.
• TIGIT expression is increased on T and NK(T) cells from cancer patients and iscorrelated to poor outcome and response to aPD1 therapy in some indications.
• iTeos developed an anti-human/cyno TIGIT blocking mAb (EOS884448) and asurrogate anti-mouse TIGIT mAb with comparable properties.
• EOS884448 properties and functionality make it an attractive Immuno-Oncologytherapy candidate:ü Strong binding to primary human and cyno T cells (sub-nM Kd)ü Competition with natural ligands with IC50 in sub-nM rangeü Increase of primary T cell functions in healthy donors and cancer patientsü Strong antitumor efficacy in monotherapy and combination with aPD(L)-1
(with surrogate mAb) in mouse CT26 and Hepa 1-6 models.
NaïveTregMemoryEffector
CD4+ T cells CD8+ T cells
TIGITTCRγ
δ
CD4 naiv
e T ce
lls
CD4 effe
ctor T
cells
CD4 mem
ory T
cells
CD4 Tre
g
CD8 naiv
e T ce
lls
CD8 effe
ctor T
cells
CD8 mem
ory T
cells
0
20
40
60
80
100
% o
f TIG
IT e
xpre
ssio
n%
TIG
IT+
cells
Fig. 1. Flow cytometry analysis of TIGIT expression, gating on different lymphocyte subsets in healthydonor (A) PBMCs (n=7). Frequency of TIGIT expressing cells is highest on CD4+ Tregs and effector andmemory T cells and (B) further increases in cancer patient PBMCs or tumor infiltrated lymphocytes (TILs)(n=12). Infiltrated Tregs display both the highest frequency and density of TIGIT receptor.
Fig. 2. EOS884448 displays a subnM affinity to primary T cells from healthy donors and cancerpatients and potently prevents binding of TIGIT ligands. (A) Human PBMCs from healthy donors andcancer patients, as well as cynomolgous PBMCs were incubated with dose escalating concentrations ofEOS884448. Kd of binding was calculated based on normalized frequency of TIGIT+CD8+ at the differentconcentrations tested. (B) EOS884448 prevents CD155 ligand binding to TIGIT overexpressing Jurkat cells,showing IC50 values at subnM range.
Fig. 3. EOS884448 potently restores pro-inflammatory cytokines in presence of CD155 ligand. (A) Humanprimary CD3+ or CD8+ T cells were stimulated with APC-like cells (CHO-TCR-CD155). Addition of aTIGIT duringstimulation prevents CD155-induced inhibition and increases IFNγ production in T cells from healthy donors andcancer patients. (B) EOS884448 restores intracellular cytokine release from frshly isolated cancer patient PBMCor infiltrated lymphocytes after an overnight stimulation with aCD3/CD28.
A. Healthy donor PBMC
B. Cancer patient PBMC
Fig. 4. EOS884448, as an hIgG1, shows preferential depletion of Tregs over memory CD4+ and CD8+ Tcells. (A). Absolute cell counts and depletion of different T cell populations expressing TIGIT is shown whenPBMCs are incubated in vitro in presence of EOS884448 for 20h and quantified by FACS. EOS884448shows a preferential depletion of Treg cells at 66.6 nM or lower concentrations in healthy donors. Similarpreferential depletion is observed in PBMC from cancer patients suffering from different solid tumor types (B).
A. CT26 syngeneic tumor model
B. Hepa1-6 syngeneic tumor model
Fig. 5. Surrogate mouse anti-TIGIT demonstrates potent isotype-dependent antitumor efficacy. Theefficacy of mouse surrogate aTIGIT mAb antagonist was evaluated in established CT26 (A) and Hepa 1-6(B) syngeneic tumors. Only aTIGIT ADCC permitting isotype, mIgG2a, significantly delays CT26 tumorgrowth in monotherapy. In combination with 10mg/kg aPD-1 mAb (clone RMP1-14; BioXcell), completetumor regression occurs in most of the animals treated with mIgG2a aTIGIT mAb.
EOS884448 is a highly potent antagonist aTIGIT mAb that:
v Binds with high affinity to human and cynomolgus TIGIT+ T cells.
v Competes for binding with TIGIT ligands.
v Increases IFNγ secretion on human primary T cells from healthy donors and
cancer patients in vitro
⇒ All at sub-nM concentrations
v Preferentially depletes human Tregs in vitro.
When replaced by a mouse surrogate with identical properties:
v Inverts the immunosuppressive Treg/CD8+ ratio and restores CD8+ T cell
IFNg secretion in vivo.
v Promotes antitumor immunity as monotherapy or in combination with
aPD-1 in CT26 colon carcinoma and Hepa 1-6 liver hepatoma mouse models.
Fig. 6. Antitumor efficacy is associated with increased effector and cytotoxic functions. (A) Flowcytometry analysis of dissociated CT26 tumors, done 1 day after the last antibody treatment, demonstratedstrong increase of IFNγ producing CD8+ T cells and of the CD8+/ Tregs ratio within tumors when treated with theaTIGIT mIgG2a isotype. (B) CT26 tumors were collected at day 18 after inoculation for Nanostring analysis ofgene expression and signature changes. Transcriptomic signature shows an increase in the cytotoxic and CD8+
T-cell scores, observed in combination of aTIGIT mIgG2a and aPD-1 treatment.
Iguchi-Manaka et al. PLoS One 2016Johnston RJ et al. Cancer Cell, 2014
Killing
12
1 drug = multiple anti-tumor mechanisms of action
1. Reactivation of immune response by• Suppressing TIGIT-mediated inhibitory signaling• Increasing ligand availibility for CD226 co-stimulatory receptor
2. Depletion of TIGIT+ highly suppressive Treg subpopulation and TIGIT+ tumor cells with ADCC-triggering isotype (FcyR dependent)
B. Cancer patient PBMC or Dissociated tumor cells (DTC)
20 25 30 35 400
50
100
Days post-tumor cell implant
% m
ice
with
tum
or <
1000
mm
3 mIgG2a iso ctrl 200 ugaTIGIT mIgG2a 200 ug aPD-1 + mIgG2a iso ctrl 200 ugaPD-1 + aTIGIT mIgG2a 200 ug
10 15 20 25 30 35
0
500
1000
1500
2000
Days post-tumor cell implant
Tum
or v
olum
e (m
m3 )
mIgG2a isotype 200 ugaTIGIT mIgG2a 200 ug aPD-1 + mIgG2a isotype 200 ugaPD-1 + aTIGIT mIgG2a 200 ug
rando : TV=45 mm
3
treatment
P<0.0001 (vs isotype)
P<0.0001 (vs PD-1)
10 15 20 25 30 35
0
500
1000
1500
2000
Days post-tumor cell implant
Tum
or v
olum
e (m
m3 )
PBS
aPD-1aTIGIT mIgG1 200 ug
aPD-1 + aTIGIT mIgG1 200 ug
rando : TV=97 mm
3
treatment
PBMC (CD3) DTC (CD4) DTC (CD8)0
20
40
60
80
% IF
Nγ+
IFNγ
PBMC (CD3) DTC (CD4) DTC (CD8)0
20
40
60
80TNFα
% T
NFα
+
PBMC (CD3) DTC (CD4) DTC (CD8)0
10
20
30
40
50IL-2
% IL
-2+
hIgG1_IsotypeEOS884448
A. Primary CD8+ T cells
B.
Med
ian
tum
or g
row
th Surv
ival
0 5 10 15 20 250
500
1000
Days post-tumor cell implant
Tum
or v
olum
e (m
m3 )
hepa 1-6 median TV
PBSaTIGIT mIgG2a
t r e a t m e n t
rando : TV=60 mm
3
p=0,0009
CT26 syngeneic tumor model
Flow
cyt
omet
ry
anal
ysis
Nan
ostri
ngsi
gnat
ures
0
20
40
60
80
100
% o
f spe
cific
lysi
s (o
n ga
ted
imm
une
popu
latio
ns) Cancer type 1
Cancer type 2Cancer type 3Cancer type 4
Total memory
Total memory
Tregs
CD4+ CD8+
B cellsCD19+
(Rituximab activity)
* *
n.s.
Kd=0.08nM
HD CD8+ CP CD8+ Cyno CD8+
0.0
0.5
1.0
1.5
2.0
Kd (n
M)
EOS884448 affinity summary
0255075
100100200300400
80012001600
Abso
lute
num
ber c
ells
/µL a-TIGIT IgG2
a-TIGIT IgG1a-TIGIT IgG4Isotype IgG2Isotype IgG1Isotype IgG4No Ab
Total Memory Tregs Total Memory
CD4+ CD8+
0
20
40
60
80
% o
f spe
cific
lysi
s (o
n ga
ted
imm
une
popu
latio
ns) Ab at 66.6nM
Ab at 0.66 nMAb at 0.0066 nMIso at 66.6nMIso at 0.666nMIso at 0.00666nM
Total Memory Tregs Total Memory
CD4+ CD8+