Antitumor efficacy of anti-TIGIT antagonist antibody ... · Iguchi-Manaka et al. PLoS One 2016...

0 2 4 6 8 10

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Antibody concentration (nM)

Norm

aliz

ed fr

eque

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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

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Days post-tumor cell implant

Tum

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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

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MFI

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155

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LGO

iTeos Therapeutics, Gosselies, Belgium Corresponding author: catherine.hoofd@iteostherapeutics.com

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

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IT e

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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

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**p=0.005 **

IC50 = 0.04nM

-3 -2 -1 0 1 2 3

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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

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CD8 mem

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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)

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rando : TV=45 mm

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treatment

P<0.0001 (vs isotype)

P<0.0001 (vs PD-1)

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aPD-1aTIGIT mIgG1 200 ug

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rando : TV=97 mm

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treatment

PBMC (CD3) DTC (CD4) DTC (CD8)0

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hIgG1_IsotypeEOS884448

A. Primary CD8+ T cells

B.

Med

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tum

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CT26 syngeneic tumor model

Flow

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anal

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Nan

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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+

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EOS884448 affinity summary

0255075

100100200300400

80012001600

Abso

lute

num

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/µL a-TIGIT IgG2

a-TIGIT IgG1a-TIGIT IgG4Isotype IgG2Isotype IgG1Isotype IgG4No Ab

Total Memory Tregs Total Memory

CD4+ CD8+

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20

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lysi

s (o

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imm

une

popu

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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+